Understanding Milk Protein Adsorption as a Model to

Study Sample Loss in Proteomics

by

Ameya Ranade

Master of Science, Northeastern University, 2013

Bachelor of Pharmacy, R.T.M. Nagpur University, 2010

Thesis Submitted in Partial Fulfillment of the

Requirements for the Degree of

Master of Science

in the

Department of Chemistry

Faculty of Science

© Ameya Ranade

SIMON FRASER UNIVERSITY

Fall 2017

Copyright in this work rests with the author. Please ensure that any reproduction or re-use is done in accordance with the relevant national copyright legislation.

ii

Approval

Name: Ameya Ranade

Degree: Master of Science

Title: Understanding Milk Protein Adsorption as a Model to Study Sample Loss in Proteomics

Examining Committee: Chair: Dr Jeffery Warren Assistant Professor

Dr Bingyun Sun Senior Supervisor Assistant Professor

Dr Vance Williams Supervisor Associate Professor

Dr Dipankar Sen Supervisor Professor and Chair

Dr Byron Gates Internal Examiner Associate Professor

Date Defended/Approved: August 25, 2017

iii

Abstract

Non-specific protein adsorption is one of the causes of sample loss in

biological experiments. This is a cause of concern in studies where samples are

complex and many of the constituent proteins are low abundant, unquantified or

unidentified. Since the proteins are irreversibly lost from the samples, it eludes their

detection and their role in biological systems cannot be ascertained. This sample loss is

unpredictable and non-reproducible which leads to distorted data. On an industrial scale,

non-specific adsorption of proteins on machinery may reduce the machine’s efficiency

and life. Similarly, unaccounted sample loss due to adsorption during storage contributes

to transmission losses to the manufacturer. Various external factors affect protein

adsorption that can be exploited to reduce sample loss. In this work, we studied milk

proteome adsorption and attempted to quantify the effect of three prominent external

factors on the differential adsorption pattern of milk proteins. For this project, we

optimized an in-house developed DPA method based on SDS-PAGE, which not only is

tag-less and MS compatible but also fast and economical.

Keywords: Non-specific protein adsorption, DPA-SDS-PAGE, sample loss, external

factors

iv

Dedication

This thesis is dedicated to family and friends who made this journey more exciting than

the destination itself.

v

Acknowledgements

I would like to thank my senior supervisor Dr Bingyun Sun for giving me an

opportunity to initiate and finish this project. She always found funds to support me and

the lab even in difficult times. I will always be indebted to her. I would also like to extend

my sincere gratitude to my committee members Dr Vance Williams and Dr Dipankar Sen

for providing insight into my data and guiding through the most difficult times of my

thesis in an endless number of the committee meetings. Without their constant support, I

would never have finished this work.

I am thankful to Ms Nathalie Fournier, graduate secretary for the chemistry

department. She always made sure that I meet all administrative deadlines throughout

the programme. She was of immense help during the last part of thesis submission. I

would also like to thank Mr Paul Mulyk for assisting me with his technical expertise and

chemistry department for providing funds and infrastructure for my research.

The graduate programme is inherently challenging and I am grateful that I had an

outstanding team from Health and Counselling Services for helping me sail through the

stormy seas. I am especially thankful to Mr Dylan LeRoy for bearing with for more than

two years, Ms Susan Brook and my collogues at Thesis Support Group for helping me to

stay grounded and pointing me to right resources when needed.

Lastly, I would like to thank my dearest friends Hitesh Arora and Lalangi

Chandrasena for being my family and strongest anchors. Everything I am today, I owe to

you. I am thankful to my collogues Dr Sean Fenwick, Xuan, Mahsa, Oluwafemi,

Adejumoke and Oluwafemi for all great times and memories that we created together.

vi

Table of Contents

Approval ............................................................................................................................... ii

Abstract ............................................................................................................................... iii

Dedication ........................................................................................................................... iv

Acknowledgements ..............................................................................................................v

Table of Contents ................................................................................................................ vi

List of Acronyms ............................................................................................................... viii

List of Tables ........................................................................................................................x

List of Figures...................................................................................................................... xi

Chapter 1. Introduction ............................................................................................... 1

1.1. Protein adsorption - definition ................................................................................... 1

1.2. Proteomics – its importance and challenges............................................................ 3

1.3. Forces involved in protein adsorption....................................................................... 4

1.4. Factors affecting protein adsorption ......................................................................... 6

1.5. The behaviour of proteins at the interface ................................................................ 9

1.5.1. Protein orientation and conformation at the surface ........................................ 9

1.6. Discussion on milk proteome .................................................................................. 11

1.7. Structure and brief discussion on chemistry of major milk proteins ...................... 12

1.8. Gel electrophoresis and direct protein analysis ..................................................... 15

Chapter 2. Methodology development – protocol optimisation........................... 18

2.1. Gel electrophoresis and direct protein analysis ..................................................... 18

2.2. Materials and instruments ...................................................................................... 19

2.3. Sample preparation ................................................................................................ 19

2.3.1. Adsorption and stripping ................................................................................. 19

2.3.2. Sample reconstitution and standard preparation ........................................... 20

SDS-PAGE ............................................................................................................... 20

2.3.3. Fixing and Staining ......................................................................................... 21

2.3.4. Optimising staining method for SDS-PAGE gels ........................................... 21

Time-dependent silver staining ................................................................................ 25

ImageJ analysis ....................................................................................................... 28

Confirming Identity of bands using mass spectrometry .......................................... 29

Using Mass Spectrometry to Quantify Proteins ...................................................... 30

Issues with Protein Quantification ........................................................................... 32

2.4. Characterization of dynamic range and sensitivity of silver staining ..................... 33

Chapter 3. Methodology development for milk adsorption studies .................... 36

3.1. Optimisation of washing efficiency ......................................................................... 36

3.2. Optimization of stripping agent ............................................................................... 37

3.2.1. Organic and inorganic stripping agents ......................................................... 39

3.2.2. Selecting SDS-NH4HCO3 ............................................................................... 40

vii

3.2.3. Reproducibility of 100 mM NH4HCO3-0.5% SDS (ABC) ................................ 42

3.3. Volume test ............................................................................................................. 44

3.4. Concentration test ................................................................................................... 45

Chapter 4. Studies on milk protein adsorption ...................................................... 48

4.1. Concentration saturation......................................................................................... 48

4.2. Time saturation ....................................................................................................... 50

4.3. Effect of temperature .............................................................................................. 54

4.4. Effect of pH ............................................................................................................. 55

4.5. Effect of surfaces .................................................................................................... 58

Chapter 5. Conclusion and future work .................................................................. 60

5.1. Conclusion .............................................................................................................. 60

5.2. Future work ............................................................................................................. 60

References ....................................................................................................................... 62

Appendix A ...................................................................................................................... 72

1. Time Dependent Silver Staining ......................................................................... 73

2. Standard curves from time dependent silver staining ..................................... 76

viii

List of Acronyms

ACN Acetonitrile

BSA Bovine Serum Albumin

LBPs Lowly Abundant Proteins

HAPs Highly Abundant Proteins

ALA α Lactalbumin

DPA Direct Protein Analysis

DTT Dithiothreitol

ELISA Enzyme-Linked Immunosorbent Assay

BLG β Lactoglobulin

FA Formic Acid

AC α Casein

HCl Hydrochloric Acid

BC β Casein

KC κ Casein

IAA Iodoacetamide

ABC 100mM Ammonium Bicarbonate 0.5% SDS

μg Micro gram

μL Microlitre

L Liter

APS Ammonium Persulphate

MS Mass Spectrometry

ix

NaOH Sodium Hydroxide

PBS Phosphate Buffered Saline

PEG Polyethyleneglycol

SDS-PAGE Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis

x

List of Tables

Table 1-1 Physiochemical constants of milk proteins with their role 40,71 ................. 14

Table 1-2 Mobility of milk proteins on SDS-PAGE 86 ................................................ 17

Table 2-1 Percent quantities of major milk proteins identified by mass spectrometry................................................................................................................... 31

Table 2-2 Comparison between reported and experimental adsorption values for major milk proteins .................................................................................... 33

xi

List of Figures

Figure 1-1 Types of protein orientations .................................................................... 10

Figure 1-2 Major casein proteins ................................................................................ 12

Figure 1-3 Major whey proteins .................................................................................. 14

Figure 2-1 Structure of Coomassie dye and Staining of Coomassie dye of milk proteins on 15% gel .................................................................................. 22

Figure 2-2 Silver Staining of 15% gel ......................................................................... 24

Figure 2-3 Sequential staining of 15% SDS gel with milk protein standards, first with Coomassie staining followed by silver staining ........................................ 25

Figure 2-4 Analysis of 1.0 μg and 0.3 μg standards with background (A) and after background substraction (B) ..................................................................... 27

Figure 2-5 Analysis of 0.1 µg standards with background (A) and after background

substraction (B) ......................................................................................... 28

Figure 2-6 Generation of Standard Curve from ImageJ output ................................. 29

Figure 2-7 Schematic representation of mass spectrometry based identification .... 30

Figure 2-8 Schematic representation of mass spectrometry based quantification ... 31

Figure 2-9 Dynamic range of milk protein with silver staining ................................... 34

Figure 3-1 Optimization of washing efficiency ........................................................... 37

Figure 3-2 Need for optimisation of new stripping agent ........................................... 39

Figure 3-3 Quantification of various chemicals for stripping efficacy ........................ 40

Figure 3-4 Quantification of various chemical mixtures for stripping efficacy ........... 42

Figure 3-5 Reproducibility of 100 mM NH4HCO3-0.5% SDS as stripping agent ....... 43

Figure 3-6 Volume test of 100 mM NH4HCO3-0.5% SDS as stripping agent ............ 45

Figure 3-7 Concentration test of 100 mM NH4HCO3-0.5% SDS as stripping agent 46

Figure 4-1 Concentration saturation of polypropylene vial surfaces at various protein concentrations ........................................................................................... 50

Figure 4-2 Time saturation of polypropylene vial surfaces at various incubation periods....................................................................................................... 53

Figure 4-3 Analysis of effect of temperature on milk protein adsorption on polypropylene ............................................................................................ 55

Figure 4-4 Quantification of the effect of pH on milk protein adsorption on polypropylene vials (Gel 1) ....................................................................... 57

Figure 4-5 Quantification of effect of pH on milk protein adsorption on polypropylene vials (Gel 2) ............................................................................................... 58

Figure 4-6 Analysis of the effect of nature of adsorbate surfaces on milk protein adsorption on polypropylene vials ............................................................ 59

1

Chapter 1. Introduction

1.1. Protein adsorption - definition

Innumerable chemical, physical and biological processes take place either on the

boundary of two phases or are initiated at that interface.1 One of the physical processes

that take place at the interface, changes the concentration of substance under

investigation in comparison to other substances at the interface.1 This process is defined

as ‘Adsorption’. Two terms that are often used while explaining adsorption, are

adsorbate and adsorbent. An adsorbent is a substance that gets adsorbed, which in this

case is protein in solution and adsorbate is the material or surface on which adsorption

takes place, which in this study is a vial surface.2,3 Protein adsorption refers to the

accumulation of proteins at the interface.4 Often the interface between the protein

solution and surface of the container is a thermodynamically unstable system. The

system can be stabilized by reducing Gibbs Free Energy at the interface. Some of the

ways to reduce this free energy from the system, include dehydration of part of proteins

or adsorbate, interactions between amino acid side chains of the proteins with adsorbent

surfaces or conformational changes of proteins. In order to reduce the Gibbs free

energy, proteins in solution adsorb spontaneously to the surface.5 Hence, protein

adsorption is a thermodynamically favourable process. However, the favourable nature

of the process depends on the surface – protein interactions.6

Protein-surface interactions are influenced by the nature of constituent amino

acids and involved intermolecular forces. The chemical nature and arrangement of

amino acid side chains create regions on protein surfaces that distinctly vary in their

charges and hydrophobicity and hydrophilicity, making proteins amphiphilic in nature. 5

Regardless of charges, protein surface can interact with variety of surfaces by

intermolecular forces such as Coulombic forces, Van der Waals forces, Lewis acid-base

forces and entropically based forces such as hydrophobic interactions, conformational

entropy and restricted mobilities.7 Protein adsorption can be classified as specific, or

non-specific depending upon the adsorption preference or affinity of the proteins towards

the adsorbate.8 This study is focused on the investigation of non-specific protein

2

adsorption, whose unpredictable nature has been the cause of concern for investigators

across all disciplines.9

Protein adsorption has biological and nonbiological importance, which is one of

the reasons that makes it a widely studied topic. For instance, non-specific adsorption of

proteins on implants can trigger an inflammation cascade leading to xenobiotic rejection,

or non-specific adsorption of protein on sensitive detection probes can interfere with

quantification of low abundant proteins in proteomics.8 In the dairy industry, non-specific

protein adsorption often causes clogs and blockages in ultracentrifuge units leading to

production losses.8 Proteins of biological importance have been studied for their

adsorption patterns such as co-adsorption, sequential adsorption or preferential

adsorption. For example, protein adsorption on various surfaces is studied to design

adsorption resistant implants.8,10 Other interests in protein adsorption include design of

immunoassays, biosensors and drug delivery systems.4

Proteins can be classified as hard, semi-hard and soft depending upon their

structural stability. β lactoglobulin, BSA and β casein proteins respectively are the

examples of each corresponding category.8 Research on protein adsorption has been

focused on studying adsorption of these isolates on various surfaces, such as glass,

plastics and stainless steel.11–13 The research focused not only on the quantification of

adsorbed proteins (see Table 2.1) but also on conformational and orientational changes

and time-dependent structural changes in proteins. In the series of experiments

conducted by Norde and Buijis, it was proved that external factors such as temperature,

pH and presence of Hofmeister series ions can significantly influence protein

adsorption.7,14 Further, Lu, on his work on whey proteins, proved the existence of ‘side-

on' and 'end-on' orientation of proteins.8,15 The researchers also proved that orientation

is governed by free energy minimum associated with protein-protein interactions.15

Extrapolating the concept, Andrade, et al. proved that similar to orientation, proteins also

have free energy dependent, and preferred conformation at the interface, which is

different from its native conformation.5 During adsorption, proteins change from their

native conformation to preferred conformation due to favourable protein – surface

interactions and gain in entropy caused by loss of ordered secondary structure and

release of counter ions or solvating molecule.5 Creighton et al, from their work,

established the slow nature of orientation cascade for proteins adsorbed at surfaces and

3

proved that proteins are more resistant to desorption after completion of orientation

cascade.7

Protein adsorption is a time-dependent process, ranging from few seconds for

BSA to few hours for milk proteins, and hence also displays kinetics.16 A review by

Rabe, et al. summarizes the kinetics of protein adsorption, which starts with the

approach of proteins to the surface and continues until all the available adsorbate

surfaces are saturated.8 Following this, the system enters a state of equilibrium, where

the amount of adsorbed proteins equal to the amount of desorbing proteins. Cuypers et

al. gave an equation to deduce adsorption and desorption rate of proteins.17 Using

similar equation Grygorczyk et al. and Mercandante, et al., gave dissociation constant,

Koff and Kon values of caseins and whey proteins.18,19 Though various theoretical models

of protein adsorption exist to predict the adsorption behaviour of proteins under given

conditions there is still a lack of sufficient data to predict and stop non-specific protein

adsorption.20,21

1.2. Proteomics – its importance and challenges

A proteome is the complete set of proteins expressed by a cell, tissue or an

organism at a given time.22 Proteomics is the high throughput study of identification and

quantification of a proteome. The composition of the proteome is dynamic and varies

with the time and stress level.23 Proteome comprises of highly abundant proteins that

make up the majority of protein mass, and a huge number of lowly abundant proteins.24

By sheer concentration, the highly abundant proteins mask the detection and elucidation

of lowly abundant proteins.25 This is a hindrance in proteomics as the role of low

abundant proteins cannot be established in a biological process.22 Since all the proteins

in the proteome are already characterized, it is convenient to compare the standard list

of identified protein to the manipulated samples to determine changes in individual

protein levels.

The adsorption process is triggered as soon as protein comes in contact with the

solid surface.10 Proteomics experiments involve the use of many different solid surfaces

such as polypropylene sample vials and pipette tips.26 Since adsorption is non-specific,

all kinds of proteins tend to adhere to these surfaces. The quantities of low abundant

proteins (LAPs) are minuscule as compared to high abundant proteins hence, even a

4

fraction of loss of proteins from soluton can push the LAPs below the detection limit of

the instrument or to be completely masked by abundant proteins.27 Adsorption of

proteins to surfaces causes irreversible loss of proteins from the sample, which

permanently alters the constitution of sample causing a negative impact on the outcome.

Identification, quantification and functional elucidation of LAPs in proteomics, will

therefore be hindered if they are lost from the sample.

Understanding how different proteins adsorb, can help in designing strategies to

minimise this non-specific adsorption. Investigating the differential adsorption spectrum

of different proteins in the proteome, helps us understand the affinity of individual

proteins to the adsorbent in a complex sample mixture.8 Protein adsorption is influenced

by the nature of protein itself, often referred to as internal factors and non-protein or

environmental factors, often described as external factors.8 It has been reported that by

manipulating the external conditions that affect adsorption such as temperature, pH and

the nature of the adsorbent surface, non-specific adsorption can be minimised.8 The

information thus obtained regarding the influence of external factors can also help us to

devise methods to prevent excessive protein adsorption.10

The purpose of this study is to investigate the impact of non-specific protein

adsorption from protein mixtures on the various surfaces routinely encountered in the

laboratory such as polypropylene, glass and adsorption resistant sample vials.

Traditionally, research on protein adsorption has focused on the study of few model

proteins or few components of the proteome in isolation.28,29 In the protein mixture, each

constituent protein is physicochemically different and therefore, they differ in their

mobility and affinities towards the adsorbent surface. This creates competition among

the protein molecules for available adsorption sites. In other words, rapid initial

adsorption of high mobility, low-affinity proteins to the surface, can be replaced by high

affinity, low mobility proteins. The change of adsorption spectrum due to this

phenomenon is called ‘Vroman Effect’, which cannot be studied in isolated systems.17,30

In proteomics, when all proteins are the concern of study, there is an emergent need to

study protein adsorption in the context of proteomics.22,24

1.3. Forces involved in protein adsorption

There are three major types of forces associated with protein adsorption:

5

Ionic interactions:

The charge on a protein molecule is the addition of charges on its individual

amino acids. The charges on the proteins are governed by the pKa of their amino acid

side chain which can range from 1.7 to 10.47, and pH of the surroundings. The net

charge results in electrophoretic migration in the physiological electric field. These

interactions are short ranged owing to the high dielectric constant of the water and only

play a dominant role when the protein approaches the surface with an opposite charge

as compared to the surface.31

Hydrogen bonding

Proteins form hydrogen bonds between the carboxylic and amide chain that

gives its secondary structure. During adsorption, proteins undergo changes in their

orientation and conformation, and in the process exchange hydrogen bonds with

surfaces. Thus, hydrogen bonding is one of the driving forces in protein adsorption.32

Hydrophobic interactions

For the majority of proteins, the three-dimensional structure is essential for its

functions. The driving force required for maintaining this three-dimensional structure

comes from intramolecular interactions between peptides, such as hydrophobic

interactions, hydrogen bonding and disulphide bridge formation, to name a few.

Hydrophobic interactions help protein to shield the nonpolar, hydrophobic amino acids

by hiding them in the core structure. This arrangement allows proteins to decrease their

surface area and thus avoid unwanted interactions with water. Gibbs energy (∆G=∆H-

T∆S<0) decreases when the hydrophobic regions of the adsorbent and the protein get

dehydrated (predominately due to an increase in entropy of the liberated water

molecules attached to protein core).33 The hydrophobicity of the protein exterior

influences protein adsorption at solid/water interfaces. In general, protein molecules

change their conformations to a large extent on hydrophobic surfaces than on

hydrophilic surfaces. This is because the hydrophobic part of the protein and the

hydrophobic part of the surface interact together.34

6

Exposure of hydrophobic amino acids to water is energetically unfavourable,

which leads to loss of system entropy.35 The system then tends to form a layer of ordered

water molecules around the protein to minimize this loss of entropy. However, the entropy

still remains negative, which translates to negative value for Gibbs Free Energy, leading to

protein adsorption.35

1.4. Factors affecting protein adsorption

Proteins are macromolecules with their domains differing in physiochemical

properties. Owing to this phenomenon, protein adsorption is affected by external

environmental factors such as temperature, pH, nature of the adsorbent surface, etc.

and by intrinsic factors such as protein size, structural stability and composition.

Following is the account of the effects of these factors:

External factors affecting protein adsorption

Milk proteome is a relatively simple and completely elucidated system. For the

reasons detailed in the upcoming section, it has been used as a model system to study

protein adsorption in this work, and hence this section primarily focuses on the

behaviour of these proteins towards changes in external factors.

Temperature

At higher temperature, milk proteins showed a remarkable increase in adsorption

amount.8,36 Also at higher temperatures, milk proteins tend to lose their native

conformation and get denatured, which also increases adsorption.8,36

The literature reported value for adsorbed β lactoglobulin at a higher

temperature ranged from 1.05 to 1.68 mg/m2 for 42 ºC and 65 ºC respectively on the

glass surface.8,18

pH

The simultaneous presence of an equal number of positive and negative charges

on a protein makes them Zwitterionic. In such a case, the net charge on the protein is

determined by the pH of the solvent in which they are dissolved. The isoelectric point is

7

a pH at which the net charge on the protein is zero. At low pH, that is when pH<pI,

proteins are positively charged and negatively charged when pH>pI. The electrostatic

repulsions are minimum at the isoelectric point, which results in denser adsorption of

proteins.8 Though adsorption increases when the proteins and surface are oppositely

charged, which results in accelerated migration of proteins to the surface, the net protein

adsorption is still maximum at the isoelectric point of proteins, since absence of charge

allows denser packing of protein at the interface whereas such close packing is not

possible due to repulsive forces that come into play when similarly charges proteins

approach each other. This phenomenon, in which adsorbing proteins interact not only

with the surface but also with one another is referred as lateral interactions. The similar

charges present on proteins impart a long range intermolecular repulsion that prevent a

close packing of proteins.8,37 Milk proteins are classified as whey and caseins due to

their distinct physiochemical properties38. Also, micelle caseins are known to stabilise

milk due to surface charge repulsion.39–41 The surface charges on proteins change with

surrounding pH, which influences changes in their adsorption pattern. Hence,

investigating role of pH in protein adsorption in also in the aegis of this study.8

Influence of adsorbent surface on protein adsorption

The nature of the adsorbent surfaces is an important parameter affecting protein

adsorption. Various parameters that influence adsorption are surface energy, polarity,

charges and morphology of the adsorbent surface.42 Protein adsorption has been

studied on various unmodified surfaces such as quartz, glass and graphite to Self-

Assembled Surfaces Monolayers (SAMs).20,43,44 Proteins tend to adsorb strongly to

nonpolar and high surface tension surfaces when compared to polar and low surface

tension surfaces8. The exception to this general rule is glycoproteins. Due to their

hydrophilic glycan side chains, which entrap the hydrophobic surfaces in the glycan

shell, the glycoproteins adsorb rapidly on to polar surfaces.8,45

The Ionic strength of dissolved ions

The concentration of dissolved ions, described by ionic strength is a contributing

factor in protein adsorption.8 Debye length is the measure of ion's net electrostatic effect

in solution and the distance to which the electrostatic effects persist.46 Higher ionic

strength corresponds to a shorter sphere of influence between charged moieties. With

8

regards to protein adsorption, at higher ionic strengths, adsorption of charged proteins to

oppositely charged adsorption surfaces is hampered and that to like-charged surfaces

enhanced. The presence of ions can change the lateral electrostatic protein-protein

interactions that are responsible for influencing packing density, initiating

inter-protein repulsions or propagating protein aggregation.15,47,48

The Hofmeister Series describes the precipitating out effect on proteins by some

ions. Ions such as sulphates, fluorides, magnesium and calcium ions stabilise the native

structure of proteins and cause them to precipitate out of the solution.8,49 Similarly, per-

chloride, thiocyanate and ammonium ions destabilise the native protein structure thus

decelerating their precipitation.8,49 For instance, caseins have been reported to have

calcium core that along with κ caseins, hold the micelles together.41 Increased in calcium

concentration in solutions has been demonstrated to stabilise the caseins structure and

reduce adsorption.8,40,50,51

Intrinsic protein properties affecting adsorption

Protein biomolecules are made up from 20 amino acids.8 These along with a

plethora of variations in the form of attached sugars and other non-protein moieties,

impart a unique complexity and diversity to these molecules.52,53 which impede

prediction of adsorption pattern of proteins.8,54

As mentioned earlier, proteins are classified as soft and hard proteins depending

upon their structural flexibility. Proteins such as β lactoglobulin, lysozyme and

α Chymotrypsin are classified as hard owing to their little flexibility with regards to a

conformational change that happens during adsorption.8,55 Proteins such as albumin,

transferrin and immunoglobulin, are examples of proteins with an intermediate

flexibility.8,56 A peculiar feature of these type of proteins domains is that they vary with

regards to charges and degree of hydro/lipophilicity. This imparts a degree of flexibility to

these proteins to undergo conformational reorientation during adsorption. Further, this

group of proteins can exist in multiple states of adsorption that vary in their adhesion

energy. The other extreme group of proteins are lipoproteins and glycoproteins.8

Adsorption properties of these proteins is governed by the lipids or glycans associated

with these proteins. For example, lipoproteins are structurally not very robust, that

makes them easy to adsorb on hydrophobic surfaces along with significant changes in

9

its conformations while doing so. Glycoproteins, on the other hand, are difficult to adsorb

on hydrophobic surfaces owing to the hydrophilic nature of its glycans.8

1.5. The behaviour of proteins at the interface

Langmuir explained the theory of adsorption using an assumption that gas

molecules are spherical and rigid.8,57 However, proteins are macromolecules that seldom

are spherical and most of the time are unsymmetrical. Due to the defined folding into

secondary and tertiary structures, proteins have distinct domains that can be classified

based on charges and hydrophilicity and hydrophobicity that distinctly influence the

adsorption pattern of proteins. Further, surface charges on protein molecules change

with change in environmental pH which, adds another dimension to the problem.8,58,59

1.5.1. Protein orientation and conformation at the surface

In bulk solution, proteins are in the state of free random motion that determines

which part of the protein will interact with the adsorbent surface, thus inducing an

orientational change in proteins. This orientation is determined by the free minimum

energy of the protein which results from interactions with the Coulomb and Van der

Waals forces and hydrogen bonds along with changes in entropy due to gain of solvent

molecules or loss of entrapped ions.8 Depending upon the chemical nature of associated

amino acid side chain, the protein surface is often divided into hydrophobic, hydrophilic

or charged surfaces in order to explain the affinity of the protein domain to the adsorbent

surface.60

10

Figure 1-1 Types of protein orientations

(Adapted from Rabe M., et al., ' Understanding protein adsorption phenomena at solid surfaces. Adv. Colloid Interface Sci')

During the initial stages of adsorption, protein-surface interactions are preferred

and mostly protein-protein interactions are not favoured leading to rapid adsorption.

However, with increased protein density at the surface, a new energy minimum is

established, due to now favoured and ever dominating protein-protein interactions. At

the initial stages of adsorption, proteins adopt an orientation to maximise their surface

area. However, with the rise of protein density at the surface, this orientation becomes

less favourable. Once a critical protein density is reached the adsorbed proteins undergo

a change of orientation by rotating the surface bound proteins, which leads to increased

desorption rate. For proteins that are rectangular (with a dimension of a*b*c), two types

of orientation are possible. One is called “end-on” with the long axis and the other is

called “side-on” with the short axis perpendicular to the surface.8 At low concentration

(0.04 mg/mL) proteins usually take on a side-on configuration.8,20,61 Especially globular

proteins, which have an ellipsoidal shape adsorb with a side-on type configuration. But a

high concentration (0.2 mg/mL) adsorption takes place in two different steps.8,20 The first

step is fast and proteins get direct adsorbed without changing their conformation. The

second step is slow and the thickness of the layer increases gradually with the adsorbed

amount. This is due to proteins changing from a side-one to an end-on type orientation,

so a higher absorbed amount can be observed.8

11

The conformation of the protein depends upon the free energy minimum, which

in the case of adsorption is not the same as energy minimum of protein in solution.

Hence, once in contact with the surface, proteins try to increase their surface footprint by

undergoing conformational changes, which allow them to cover maximum surface area

possible. This is possible on account of increases in free energy.17,48 The extent of

conformational changes depends upon the flexibility of the protein. The conformation at

the surface is often not the native state or conformation of the protein. Proteins have a

preferred conformation similar to a preferred orientation.8

Proteins approach the surface from bulk solution in their native form. After the

initial contact with the surface, there is an increase in the protein-surface interaction that

causes the protein to lose its ordered secondary and tertiary structure along with the loss

of solvent molecule or counter ions leading to entropy gain that promotes adsorption, by

the loss of free energy.8,28,44 During this process protein relaxes wherein it tries to

maximise its surface area leading to resistance to elution or being washed off. The

change in conformation is often a very slow process that can take days to be completed.

Many proteins initially bind to the surface with low binding energy, which significantly

increases with time as the protein has undergone changes in conformation. This

phenomenon explains the observation that freshly adsorbed protein layer is least

resistant to elution as compared older protein layer which may be irreversibly attached to

the surfaces.8

1.6. Discussion on milk proteome

Milk has been selected as the source of protein for this study. Milk is the

commonly used protein in various experiments as a blocking agent in various

biochemical experiments. About 500 proteins have been identified in milk up to this

date.62 Previous research on milk proteome adsorption had focused only on studying an

isolated component, either as an individual protein or a class of proteins at a time.

Hence, though we understand kinetic and thermodynamic models of adsorption of these

isolates, we are still missing the holistic picture. In a mixture of proteins, initially, highly

abundant and low-affinity proteins are adsorbed first and with the passage of time, these

proteins are replaced by low abundant, high-affinity proteins, a phenomenon known as

'Vroman Effect'.17 Hence, our group is more interested in studying the adsorption

spectrum of the major milk proteins across the whey and casein classes.

12

1.7. Structure and brief discussion on chemistry of major milk proteins

Milk is a complex emulsion of proteins, carbohydrates, fats, water, etc. Each

100 g of milk contains 87.8 g of water, 4.8 g of carbohydrates, 3.9 g of fats, 3.2 g of

proteins, 120 mg of calcium and 14mg of cholesterol.63 Milk proteins primarily have two

types, Caseins and Whey proteins. Majority of caseins are poorly soluble in water while

the whey proteins are soluble in water.64,38,65,66

Figure 1-2 Major casein proteins

Caseins constitute 80% of the milk proteins and have α-s1, α-s2, β and κ

subtypes.40. Caseins exist as micelles in water. Caseins are phosphoproteins with fairly

large number of serine residues. The α caseins consist 8-10 seryl groups while β

caseins have five phosphoserine residues, and is more hydrophobic than α and κ

caseins.40 As

α and β caseins are phosphorylated, they are very sensitive to the presence of calcium

ions.66–68 These caseins are precipitated under high concentration of calcium ions.68 The

κ caseins, on the other hand, are glycosylated and have one phosphoserine residue and

hence stable in the presence of calcium ions.40 The major role of κ caseins is to prevent

other caseins from precipitating and to stabilise the micelles.40,41 With an exception to

κ caseins, caseins are characterised by the absence of disulphide linkages.38 Caseins

are not heat sensitive. Their solubility decreases with increase in temperature above

120 ºC, but they are sensitive to pH and readily precipitate at isoelectric point.41 Casein

micelles are spherical with a diameter ranging from 700 to 2800 angstroms and

composed of a hydrated core surrounded by casein proteins.41 The micelles are

stabilised by the interaction of proteins with calcium ions. The κ caseins form the

outermost hydrophobic layer of the micelles. This layer is negatively charged and repels

13

other like charged micelles, thus maintaining the colloidal nature of milk. With regards to

the conformation of casein, there are three models that explain this phenomenon. All the

three models though, consider caseins colloidal particles.41

Coat core model – The model depicts the formation of low weight ratio

complexes α and κ caseins in absence of calcium. The α and β monomers with charged

phosphate loop begin to aggregate to a limiting size cassinate core.41 The formation of

core stops when a mono-layer of the α-κ complex is formed. The cassinate core has

evenly distributed κ caseins on the surface. The size of the complex is determined by the

concentration of the κ casein.68

Sub-micelle model – Proposed by Walstra in 1984, the model suggests that

micelles are made up of spherical subunits.69 Each subunit is 20-25 nm in diameter and

consists of 20 casein molecules. The subunits are kept together by hydrophobic

interactions between proteins and calcium linkages41,68 Primarily, there are two types of

sub-micelles; the α- β micelles that have a hydrophobic core and the α-κ caseins that are

hydrophilic due to sugar residues. The κ caseins form a hat-like protrusion on the

surface that prohibits further aggregation of sub-micelles by stearic and electrostatic

repulsion.

Internal structure model - Proposed by Rose in 1969, the model assumes that

β casein monomers undergo self-assembly to form chain-like polymers.70 Afterwards, α-

s1 are attached to β caseins and κ caseins attach to the α-s1 molecules forming an

aggregate. The casein micelles structure thus formed is stabilised by colloidal calcium

phosphate.41,68 The micelles are oriented in a way that β caseins are oriented towards

the interior of the micelles and κ caseins face towards the exterior. Physiochemical

properties and role of caseins vary depending upon the subtype.41

14

Table 1-1 Physiochemical constants of milk proteins with their role 40,71

Subtype Mol Wt PI Role

α-s1 22,068 -23,724 4.20 – 4.76 Antioxidant and scavenging, Transport of casein from ER to Golgi body

α-s2 25,230 - Antibacterial activity against E. coli and S. carnosis

β 23,944 – 24,092 4.6 – 5.1 Source of casomorphin that exhibits opioid binding to opioid receptors

κ 19,007 – 19,039 10.5 Serves as interface between hydrophobic interior and aqueous exterior of micelle

Whey proteins are globular proteins; the spherical structure being attributed to

their tertiary structure.38 The structure is characterised by bonding of hydrophobic amino

acids to the interior of the core while the hydrophilic amino acids bind to the exterior of

the core.66,71 The presence of hydrophilic amino acids to the exterior is the reason for

solubility of these proteins in polar solvents due dipole-dipole interactions.71 Globular

proteins are less stable due to the small energy difference between the folded and

unfolded state.1,66 Whey proteins are primarily made up of β lactoglobulin (~65%) and α

lactalbumin (~25%).39 Other constituent includes bovine serum albumin (~8%) and

immunoglobulin (~2%). The whey proteins make up 20% of the total protein content in

milk. The solubility of whey proteins is high in water and is pH independent.39

Figure 1-3 Major whey proteins

Bovine β Lactoglobulin is a small protein with 162 residues.39 It has a molecular

weight of 18.4 kDa and it structurally is dimeric under physiological conditions. The

protein, however, breaks down to its monomeric units at environmental pH 3 or less.72

Further, when subjected to high temperature and low pH the protein solution turns into a

gel with the protein forming long stiff strands.73 The aggregation is possible owing to a

free thiol group on the protein that serves as starting point of this reaction. The role of

Lactoglobulin is not clear, but indicates its role in active transport of bulky groups.74

15

α Lactalbumin has a molecular weight of 14.19 kDa.39 It has isoelectric point in

the range of 4.2 to 4.5.38 Unlike β Lactoglobulin, α Lactalbumin lacks the free thiol group

and hence does not form a gel when subjected to harsh conditions.75 α Lactalbumin

increases the production of lactose on stimulation by prolactin hormone, by transferring

the galactose moiety and forming β1, 4 glycosidic linkages with glucose in presence of

lactose synthase.38,71 A multimer of this protein also binds to calcium and zinc ions and

the complex has antibacterial and antitumor activity. However, a folded variant may

induce apoptosis in immature cells.76

1.8. Gel electrophoresis and direct protein analysis

Various methods such as quartz crystal microbalance77, ellipsometer78 and

neutron reflectometry79 have been investigated for the quantification of adsorbed

proteins. Except for the neutron reflectometry, the other two techniques are not very

suitable for absolute quantification of adsorbed proteins. Quartz crystal microbalance,

during quantification of adsorbed proteins also includes water molecules associated or

trapped within proteins, which affects the determination of absolute protein quantity.8,80

The solvent layer interferes with the adsorbed proteins and thus give an error when

ellipsometer is used to quantify adsorbed proteins.15 Neutron reflectometry though a very

precise method for detection of adsorbed proteins relies on a source of neutrons for

quantification. The source is very expensive and not universally present which limits its

use.16,79 Literature has also reported use of labelling techniques for adsorbed protein

quantification, such as fluorescent labelling, ELISA and radioactive labelling.12,13,61,81–83

However, such a technique involves 'tagging' the protein of interest with a fluorophore or

radioactive tag which essentially destroys the native state of proteins, thus making them

more susceptible to adsorption, resulting in different adsorption quantities than expected

from native proteins.9,83 There are, however, few techniques that can rapidly quantify

adsorbed proteins.10

Electrophoresis is an analytical technique that separates proteins based on their

molecular weights. Our group has exploited this simple, fast and inexpensive technique

to quantify adsorbed proteins. Varmette et al. used gel electrophoresis and postulated

'Solution depletion of SDS-PAGE' technique as 'label-free technique' for protein

quantification.12 These methods quantify the change concentration of supernatant

protein solution, before and after incubation to estimate the quantity of adsorbed

16

proteins. In order to demonstrate an observable change in the concentration of

supernatant, these methods need high surface area such as beads, powders or

nanomaterials16,84 and hence cannot be used in proteomics, because of small sample

size. Our group has modified this method to quantify protein adsorption in small sample

size. We call this technique as Direct Protein Analysis, wherein we concentrate the

adsorbed proteins by freeze drying them. This step increases the concentration of

adsorbed proteins above the detectable limits of

SDS-PAGE.

Milk proteins have two classes of proteins, viz.- caseins and whey. Caseins make

about 80% of milk proteome while whey contributes to 20% of milk protein mass. The

molecular weight of milk proteins varies from 14 kDa to 161 kDa. SDS-PAGE rapidly

separates and on including a series of internal standards quantifies milk adsorbed milk

proteins.10

The resolution of an individual class of proteins in the mixture depends upon the

concentration of acrylamide in the gel. Higher concentration (12%) of acrylamide gives

better resolution for whey proteins while lower acrylamide concentration (8%) resolves

caseins better. In a study conducted by Ng-Kwai-Hang, et al., α lactalbumin have the

highest mobility, followed by β lactoglobulin, α caseins, β caseins, k caseins, BSA and

immunoglobulins.85 In the experiment, the authors carried out two experiments, in the

first experiment only caseins were characterised and quantified and in a separate

experiment, whey proteins were quantified.85 The overall densitometry reading of gels

shows the sequence of protein separation. The table alongside shows the relative

percentage of each type of proteins.

17

Table 1-2 Mobility of milk proteins on SDS-PAGE 85

Highest mobility in resolving gel4,5 Average Molecular weight

Relative percentage

α lactalbumin 14300 2-5

β lactoglobulin 18400 7-12

K-casein ~19000 8-15

β caseins ~24000 25-35

α-S1 casein ~23500 45-55

α-S2 casein ~23500

Immunoglobulin G ~55000 1.2-3.3

Bovine Serum Albumin 67000 0.7-1.3

Lactoferrin 86000 0.01

The DPA technique thus offers an inexpensive and rapid alternative to explore

differential adsorption spectrum of the proteome. The work henceforth will describe

steps taken to develop this technique, which was essentially used to quantify only one

type of adsorbed proteins to one that can quantify milk proteome. The work will further

continue to use this technique to investigate the role of external factors on milk protein

adsorption.

18

Chapter 2. Methodology development – protocol optimisation

2.1. Gel electrophoresis and direct protein analysis

Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) is a

routinely used separation technique for proteins and nucleic acids.86 Glycine-SDS is

specifically used for separation of proteins.87 Though routinely used as separation

technique, on the incorporation of internal standards, the method can be used for rapid

quantification of separated proteins.10 The range of SDS-PAGE separation ranges from

5 kDa to 150 kDa, depending upon the concentration of two constituent monomers in the

gel matrix – acrylamide and bisacrylamide.16,88 Higher concentration (T%) of these

monomers results in smaller pore size of the gel matrix thus enabling better separation

of lower molecular weight proteins, and vice-versa when monomer concentration is

reduced. A unique feature of the SDS-PAGE technique is that it allows the usage of the

discontinuous buffer system. This enables larger sample volumes to be loaded on the

gel and ensures that the protein samples are stacked in a thin band, by the time they

reach resolving gel. The SDS is an anionic detergent and forms micelle with 70-80 SDS

molecules with carbon chain at the core and sulphate group at the interface. These

micelles form complexes with proteins in a fixed ratio of 1.4 μg of SDS per μg of

protein.89 Owing to the anionic nature of SDS, these SDS-Protein complexes are also

negatively charged. Due to their negative charges, these complexes migrate towards the

anode at the bottom of the gel. In course of this migration, the complex travels through

the pore size of the glycine - SDS gel matrix and are separated according to their

molecular weights.

The SDS-PAGE has been reported to be useful in quantification of adsorbed

proteins using solution depletion method, which relies on protein adsorption on larger

surface areas such as beads, powders or nanoparticles.16 To extend this method to

flatter surfaces such as vial surface, our lab developed ‘Direct Protein Analysis’ method

that quantified tightly adsorbed protein molecules directly from the vial surface. Prior, to

the development of this method, such direct quantification was not possible as the

quantification of the very low amount of proteins desorbed into high volumes of stripping

19

solution rendered the concentration below the detection limit of most of the analytical

techniques. We overcame this problem by introducing a concentration step of the

stripping solution prior to SDS-PAGE analysis. Thus, this method, without using any

state of the art instruments, allowed us to rapidly separate and quantify the proteins

adsorbed on the vial surface. This allowed us to study the differential adsorption

spectrum of a mixture of proteins. However, before using this technique to quantify the

external factors affecting milk protein adsorption, it is imperative to validate this

technique. The quantification can change dramatically depending upon the nature of

proteins to study, incubation time and concentration, nature of stripping agent, etc.

Hence, the technique had to be optimized for the various experimental parameters

before quantifying the external parameters.

2.2. Materials and instruments

Skimmed Milk powder was procured from Saputo Inc. (Montreal, Quebec,

Canada) and Sigma Aldrich (Lot – SLBK7361V, Toronto, Canada), Resolving gel buffer,

stacking gel buffer, Gel electrophoresis assembly, 30% acrylamide and ammonium

persulphate were purchased from Bio-Rad (Hercules, California, United States of

America). Polypropylene vials were obtained from VWR Inc. (Lot – 494340Z23174,

Radnor, Pennsylvania, United States of America) and Froggabio Inc. (Lot – 111000,

Toronto, Canada), Sodium dodecyl sulphate, bromophenol blue and silver nitrate were

from Sigma Inc. (St. Louis, Missouri, United States of America). The rest of chemicals

and Speed-Vac system were obtained from Thermo Fisher Scientific (Waltham,

Massachusetts, United States of America).

2.3. Sample preparation

2.3.1. Adsorption and stripping

In a typical procedure, milk-protein working solution at 50 ng/μL in PBS was

prepared by direct dilution of a stock at ~ 10 μg/μL concentration (measured by

Nanodrop adsorption at 280 nm) in PBS.10 Based on the previously described

procedure, 100 μL of the working solution was incubated with 0.65 mL Eppendorf vials

(VWR Lot – 494340Z23174) in an upright position at room temperature for a defined

time without agitation.10 After incubation, the protein solution was removed, and the

20

sample vials were rinsed by 100 μL of PBS by gentle pipetting 10 times. After discarding

this wash the sample vials were subsequently washed twice with 100 μL of PBS followed

by 15 seconds of vortex after each wash, prior to stripping. These washes ensured that

lightly adsorbed proteins were washed off prior to quantification. The remaining

adsorbed proteins on the vial surface were incubated with 100 μL of stripping agent

(0.5% SDS and 100 mM NH4HCO3 was used if not specified otherwise) at room

temperature for 15 mins.10 The obtained stripping solution was dried by speed-vac

(Thermo Scientific) immediately after for SDS-PAGE analysis.

2.3.2. Sample reconstitution and standard preparation

The dried samples were reconstituted in 10 μL of loading buffer, 3 μL of 1 M DTT

and 17 μL of water. A four-fold concentration of loading buffer consists of 0.25 M Tris

buffer, 0.02% Bromophenol Blue, 50% Glycerol and 10% SDS. The samples were

warmed in water bath until loading on the gel to prevent freezing of glycerol at room

temperature (17.8°C).

Standards were prepared by a similar procedure as samples. Standards

solutions of 0.25 μg/μL and 0.025 μg/μL were made from working stock solution. The

required amount of milk proteins was taken from standard solutions to make desired

quantities of protein standards, to which 3 μL of 1 M DTT and 10 μL of loading buffer

was added.

SDS-PAGE

Gel electrophoresis was carried out on a 15 well, 15% SDS resolving gel and

4% stacking gel as per established protocol. The capacity of each well is around 40 μL.

Care was taken to ensure that the volume of samples and standards in each well does

not exceed 30 μL to avoid spilling in the neighbouring well. The gels were run at 75 V till

the samples and standards crossed over to resolving gel, after which the voltage was

increased to 200 V and continued to run until the dye front reached the bottom of

resolving gel.

21

2.3.3. Fixing and Staining

Following the gel electrophoresis, the gels were rinsed with distilled water to

wash off excess SDS from the gel and then were incubated for 1 hour in fixer solution

comprising of 10% acetic acid, 25% 2-propanol and 65% distilled water. Fixing ensured

forging of bonds between the gel and proteins so that the later do not leach out of the gel

during subsequent steps. After fixing the gels were thoroughly washed for 2 hours in

distilled water, to rinse off all the excess acetic acid. The gels were then sensitised by

incubating in a solution of 0.02% sodium thiosulphate for 2 minutes followed by water

rinsing for

2.5 minutes with a change in water very 30 seconds. In the next step, the gels were

incubated in a solution of 0.1% silver nitrate to induce silver impregnation, for 30 minutes

at room temperature with gentle shaking. The gels were again washed with water for 2

minutes with a change of water every 30 seconds. The gels were then developed in a

solution of 2% sodium carbonate, 0.05% formaldehyde and 0.02% sodium thiosulphate,

until all the expected bands, became visible and the contrast was within an acceptable

range, following which the reaction was terminated by 5% acetic acid. The alkaline silver

staining process as discussed below is pH sensitive and only works in alkaline

environment. Changing pH to acidic medium using acetic acid, terminates the reaction.

2.3.4. Optimising staining method for SDS-PAGE gels

After gel electrophoresis, the proteins though separated were invisible and hence

the gels had to be stained for visualisation of protein bands and quantification. From,

variously reported staining techniques for SDS-PAGE, the Coomassie Blue and Silver

Staining are the most commonly used.

Coomassie Blue technique is based on the adsorption of stain Coomassie on the

protein. Coomassie dye in solution exists as cationic, neutral or anionic species. The

neutral form forges hydrophobic interactions with phenylalanine and tryptophan and

forges electrostatic interaction between the sulfonic group of the dye and arginine and to

some extent with lysine and histidine.90 The anionic species primarily forges electrostatic

interaction between its sulfonyl group and arginine, lysine and histidine amino acids of

the protein. The cationic species do not part take in the staining mechanism.91 The gel is

first incubated in a solution of Coomassie dye, acetic acid and methanol (Staining

22

Solution). This causes the entire gel to turn blue as the dye adsorbs uniformly

throughout the gel, albeit with varying affinities for the protein and non-protein part of the

gel. Following this, the gel was incubated and subjected to gently shaking in the solution

of acetic acid, methanol and water (Destaining Solution). This caused the loosely

adsorbed dye from the non-protein part of the gel to desorb, while due to the higher

affinity of the dye to the proteins, the dye adsorbed on the proteins remained tightly

adsorbed. This caused the non-protein part of the gel (background) to become fainter

than the protein band part of the gel. The contrast was controlled by varying the shaking

and introduction of fresh aliquots of the de-staining solution. A salient feature of this stain

is that it maintains linearity with Beer-Lambert’s law for more than three-fold dilutions, a

feat not achieved by silver stain.92 This method was fast, mass spectrometry compatible

and required little skill and most importantly was reversible. However, the detection limit

of this technique is 100 ng of proteins, which limits its application to study of high

abundant proteins.

Figure 2-1 Structure of Coomassie dye and Staining of Coomassie dye of milk proteins on 15% gel

On the gel from left to right, 2 μg, 1 μg, 0.5 μg, 0.25 μg and 0.1 μg of milk protein standards

Silver staining technique works on the principle of autocatalytic reduction of silver

ions to elemental silver on the proteins.93–96 The process is very similar to the

development of photographic film. Though the exact mechanism of this stain is still a

matter of confusion and debate, the process involves three major steps – sensitization of

23

gel by sodium thiosulphate, incubation with silver nitrate solution, image development by

sodium carbonate in presence of formaldehyde and sodium thiosulphate and termination

of reaction by the acetic acid solution. After each of the steps, there is an intermittent

washing step lasting for a few minutes to wash off the excess of the reagents before

proceeding to the next step. Sodium thiosulphate sensitises the gel for the reduction of

silver by forming a thiosulphate-protein complex that can attract silver ions to the protein

bands. It is this stage that is also responsible for controlling the contrast between the

protein bands and the background and hence must be meticulously controlled. Excess

incubation at this stage or inadequate washing after incubation can result in the intense

brown background that can mask the protein bands. Following this step, the gel is

impregnated with silver ions by incubating it in silver nitrate solution. The silver ions in

the solution are present either as free form or complexed to acrylamide gel or complexed

to proteins. The silver-acrylamide complex is weaker as compared to silver-protein

complex. The silver-protein complex is strong due to either salt formation between -COO

group of Aspartic or Glutamic acid or due to complexation through nucleophilic groups

such as imidazole of Histidine,

-SH group of Cystine, SCH3 group of Methionine or NH2 group of Lysine.97 The wash

following this step has to be closely monitored and time as excess wash may cause

silver ions to wash off leading to reduced sensitivity. Following this step is a

development stage carried out by sodium carbonate in the presence of weak developing

agent, formaldehyde and sodium sulphate that forms strong complex from the silver ions

leaching out of the background, which makes them unavailable for reduction.

Formaldehyde creates a latent image of the protein band, which serves as a ‘site’ for

reduction of silver ions to elemental silver for other silver ions. The colour of the band or

specifically the shade of the band is governed by the size of the silver grain formed at

the site, which is turn is dictated by the speed of reduction.98 Faster reduction leads to

the formation of bigger silver grain leading to the darker colour band.93 The detection

limit of silver stain is 100 fold higher as compared to Coomassie blue, about 1 ng of

proteins, however, the stain limits linearity only up to 10-fold standard quantity.92 This

technique is slow, requires skill and is irreversible. However, given to its higher

sensitivity, which was needed to visualize differential adsorption spectrum of milk

proteome and low abundant proteins, the silver stain was selected as the stain of choice

for this study.

24

Figure 2-2 Silver Staining of 15% gel

On the gel from left to right, 2 μg, 1 μg, 0.5 μg, 0.25 μg and 0.1 μg of milk protein standards

However, none of the staining techniques are universal. Techniques bias

changes with the chemical nature of the proteins involved. With regards to milk proteins,

Coomassie stains casein proteins better, but fails to stain whey proteins effectively as

shown in

Figure 2.1. On the other hand, the silver stain is biased towards whey proteins, while

some of the constituent milk proteins are not very effectively stained by either of the

techniques shown in Figure 2.2. To counter this problem, we also investigated,

sequential staining with Coomassie stain followed by silver staining. This allowed us to

visualize few bands that were barely visible with individual stains as shown in Figure 2.3.

This technique was not regularly used in this project, but only used to during band

isolation for mass spectrometry, to prevent the introduction of unwanted proteins into the

mass spectrometry samples.

25

Figure 2-3 Sequential staining of 15% SDS gel with milk protein standards, first with Coomassie staining followed by silver staining

Time-dependent silver staining Silver staining though a routinely used

technique for identification of low abundant proteins in a complex sample, its use as a

quantitative stain presents its own problem. The silver stain has a very high sensitivity, it

can detect protein signal as low as 1 ng, however, its dynamic range is only 10-fold as

compared to three orders of magnitude for Coomassie stain.92 After the 10-fold range,

the stain starts to get saturated.92,99 This saturation becomes more intense with time.

Silver staining of proteins is based on the autocatalytic reduction of silver ions to

elemental silver. This reaction is rapid and hence the SDS-PAGE gel must be sensitized

to this rapid change. An important step in this process is the use of sodium thiosulphate

as a sensitizing agent.94,100 Incubation with a sensitising agent must be very precisely

controlled. Under ideal conditions, with proper sensitization, SDS-PAGE would give an

intense stain for protein and a very light stain for the background. However, this is not

always the case, a few seconds of delay with sodium thiosulphate results in the intense

background which completely masks the signal from proteins. The background

successively becomes darker with the passage of time (in a matter of 20 minutes).

The cornerstone of this project was to study differential adsorption spectrum of

milk proteins and try to quantify changes in major protein bands, with regards to their

exposure to various external factors. Since this change is minuscule, we had to choose

silver stain instead of Coomassie. It was already observed that silver stain was biased

towards whey proteins and β casein as compared to other caseins, which meant that gel

26

had to be stained longer to see all casein bands. β lactoglobulin and β casein bands

always were the first to develop followed by BSA and α lactalbumin. The α casein, which

is the most abundant milk protein, and equally resistant to silver stain, appeared last, if

at all it did. By the time all the bands appeared, the ever continuously developing

background would start interfering with protein signal. By the time α casein appeared,

background interference would completely mask its signal. Hence, the signal from α

casein was not observed very regularly.

Apart from this, it also observed that the β casein bands stained rapidly and lost

linearity between the three standards fast by the time other bands had completely

stained the first two standards of β casein would have same intensity due to over

staining that would create a problem for quantification.

To resolve the above problems, the entire staining process was video recorded,

and then frames were selected for quantification. A short protocol was developed to

ensure that same area was quantified as area under the curve (AUC) across all the

bands and gels. Since each gel developed at the different time, three gels that were the

closest match with each other with regards to their staining time were selected. Since it

is difficult to compare intensities across three gels frame by frame, we captured frames

for all three gel videos at an arbitrarily selected time point of 1 minute each. Due to their

varying staining time, the intensities at selected time points did not match very well with

each other, resulting in large error bars. 1.0 µg, 0.3 µg and 0.1 µg of proteins were

routinely used as standards in experiments out of which 1.0 µg and

0.3 µg were focused on, since these standards were the ones to saturate.

As seen in the alongside Figure 2.4, the β casein appeared to saturate at least

3-time points before all the other bands and α casein band appeared at the last and had

the least intensity of all bands, which made it very difficult to quantify. It was also evident

from the data analysis that, by that time the background interference, made

quantification impossible. And no other band had saturated except for the β casein.

Subtracting the background intensity (Panel B, Figure 2.4) didn’t significantly improve

the α casein resolution, as the signal was very difficult for ImageJ to differentiate from

the background. The signal from other proteins was already intense as compared to

background and hence subtracting background, did not change their relative intensities.

27

Figure 2-4 Analysis of 1.0 μg and 0.3 μg standards with background (A) and after background substraction (B)

The β casein standards saturate with increase in staining time

The 0.1 µg standard did not saturate throughout the time of staining. The figure

28

below shows that intensity β casein kept on increasing till the end.

Figure 2-5 Analysis of 0.1 µg standards with background (A) and after

background substraction (B)

One way to resolve this problem was to quantify β casein before its standards

lose linearity and then let the staining progress for the other bands. The standard plot

thus created was used to check the quantity of β casein in milk standards and same was

matched against literature reported value. Since the values fairly matched, the method

was concluded as successful.

The α casein could only be seen when the background was very low. We tried

various techniques but we were unable to control the background interference. In other

cases, α casein bands did not appear at all. This can be attributed to the silver staining

bias, in which case the quantitation could not be carried out. Though the standard curve

could be calibrated using the mass spectrometry ratio since the adsorption spectrum

would be different that the standard protein spectrum, it was imperative that the α casein

signal is seen for ImageJ analysis. Quantification of other milk proteins by ignoring

the α casein band was not an option either. Since that would have led to over estimation

of all non- α casein proteins.

However, since α casein is most abundant protein, and Coomassie sensitive to

caseins if needed the α casein could be quantified by staining the gel with Coomassie,

followed by silver stain.

ImageJ analysis The stained gel images are digitised by a laser scanner

(Canon LiDE 110) and analysed by ImageJ (http://rsb.info.nih.gov/ij/). The protein

quantity in each gel band of the milk standard is estimated by the percentage of

individual band intensity over the total protein band intensity. A standard curve so

obtained is used to calculate the protein quantity in the sample bands of the gel.

29

Figure 2-6 Generation of Standard Curve from ImageJ output

Confirming Identity of bands using mass spectrometry

An easiest way to identify protein bands on SDS-PAGE gel, is to

incorporate a protein ladder on the gel. The protein ladder is a mixture of proteins whose

molecular weights are known. Comparing the position of the sample protein bands with

the corresponding horizontal protein ladder signal, provides a good estimation about the

molecular weight of the sample, which when compared to protein databases, can help in

identifying the compounds. Using this principle, we identified major protein bands as

BSA, Caseins, β lactoglobulin and α lactalbumin.

However, to confirm their identity beyond doubt, we used mass

spectrometry.101,102 After staining of the SDS-PAGE gel, a representative sample band

from each horizontal band was cut out. The sample was cut in 1mm X 1mm squares and

then subjected to cycles of water and acetonitrile washes till all the dye on them was

washed off and the gel pieces appear opaque white. The gels are then incubated

suceessively in reducing and alkylating solution. Following this, the gel pieces were

subjected to trypsin digestion protocol to break the proteins to smaller peptides. An

estimate of concentration of the digested proteins was made using nanodrop apparatus,

following which the samples were loaded to mass spectrometry. After the run the results

30

were compared with protein database to establish identity of the proteins.101

Figure 2-7 Schematic representation of mass spectrometry based identification

Using Mass Spectrometry to Quantify Proteins (This work

was done by my collegue Rustam Mukhtarov,) A variation of the above method was also

employed to estimate the quantities of the constituent milk proteins. Milk protein was

dissolved in denaturation solution followed by reduction and alkylation.The samples then

were incubated with trypsin followed by MCX cleaning. Prior loading on LC-MS, the

freeze dried samples were reconstituted in loading buffer and tested for protein presence

by nanodrop.

31

Figure 2-8 Schematic representation of mass spectrometry based quantification

In contrast to the previous method, we directly used adsorbed milk protein samples

instead of first subjecting them to gel electrophoresis. If done after SDS-PAGE the

digested proteins can readily loaded on the mass spectrometry as the SDS-PAGE

cleans the sample due to its matrix. However, in absence of this step and additional

‘clean-up’ step must be incorporated in the protocol to prevent the HPLC columns

getting clogged from the debris. The data from this protocol not only identified many

proteins but also helped us to establish as internal ratios of the constituent proteins in

the milk proteome. This information also opened an alternate to quantify protein

adsorption by acting as standard curves across the proteome.38,85

Table 2-1 Percent quantities of major milk proteins identified by mass spectrometry

Milk Proteins Literature Percent Detected Percent from MS

α-S1-Casein 45 to 55 21.973

α-S2-Casein 5.531

β Casein 25-35 17.040

κ Casein 8 to 15 5.531

BSA 0.7 to 1.3 2.990

β Lactoglobulin 7 to 12 9.118

α Lactalbumin 2 to 5 4.634

32

Issues with Protein Quantification Quantification of proteins on with silver

staining on SDS-PAGE proved to be difficult. This was partly due to 10 fold dynamic

range of silver stain and semi-quantitative nature of SDS-PAGE.92 Nonetheless,

quantification of whey proteins was easier and easily reproducible due to favourable

silver stain bias towards it. With regards to casein, β casein was stained most

prominently by silver stain while α casein was stained the least.

Κ casein was not seen on the gel due to its low concentration in milk. For quantification

of whey and α casein, standard plots of individual proteins from milk standards were

used. Separate β casein standards were run on the same gel for β casein quantification.

Due to staining variation, no two can ever be exactly same. Owing to this, instead of

considering absolute quantities, a holistic data that considers calculated quantities and

its statistical treatments such as standard deviation and Coefficient of Variance would be

a better estimation of adsorbed proteins. Due to background interference during α casein

quantification, this band was quantified only when the band was clearly visible and the

gel had the low background. In other cases, only β casein band was quantified. The

quantification was also cross-checked by comparing it with standard milk protein ratios

obtained from mass spectrometric analysis of milk standard by the method described by

Darling et al, it was observed that the quantitation obtained by both the methods were in

agreement with each other.103 Adsorption being a surface phenomenon, literature often

report adsorbed proteins quantities as a function of quantity adsorbed per unit area of

surface.8,104 On converting the literature reported values for milk protein adsorption to

match surface area of my vials, it was observed that the values did differ considerably.

However, this variation was due to the different protein-adsorbate pair used by other

investigators.7,105 Apart from this, the choice for source of milk protein used in other

studies such as type of milk (whole, skim, partially skim, reconstituted, species), genetic

variation of cows (Jersey, Houston, Angus, etc.), sample size (selective whey, selective

casein, most abundant, etc.) and method of analysis used, also contributes to variation

in reported results.8,39,69,70,106 However, our results that reflected properties of proteins

such as adsorption changes at CMC with regards to orientation, were in agreement with

the literature reported data.8 As discussed in Chapter 1, the change of orientation from

side-on to end-on occurs as the concentration of adsorbed proteins approaches CMC,

which for most of the individual milk proteins is 0.2 μg/μL.8,20 In our study on surface

saturation, there was a noticeable increase in adsorbed protein quantity at concentration

33

0.5 μg/μL of total protein concentration. This increase can be attributed to ‘more free

space available’ as the protein shift the orientation.

Table 2-2 Comparison between reported and experimental adsorption values for major milk proteins

Protein Adsorbed Quantity (ng)

Literature Reported12,16,29,107,108

Experimentally Observed

Bovine Serum Albumin 500 300

Caseins 900 400

β Lactoglobulin 300 400

α Lactalbumin 100 400

2.4. Characterization of dynamic range and sensitivity of silver staining

Dynamic range is the greatest linear response of detector divided by noise

detected. In my case at one extreme is the lowest detectable protein standard and at

other end is the highest protein standard that can be used, short of saturating the gel. In

SDS-PAGE these values are governed by the staining technique used. Silver stain tends

to saturate the gel at higher quantities of proteins. A larger dynamic range of standards

translates to better linearity among the standards, leading to reproducible and accurate

quantification of samples. Sensitivity refers to lowest, reproducible signal detected by

silver stain.

A series of milk proteins in duplicate, from 500 pg to 5 μg was loaded on

SDS-PAGE and subjected to gel electrophoresis, followed by fixing and staining with

silver stain. The results were analysed with ImageJ software and the lowest detectable

protein signal was identified and dynamic range was determined from that point onwards

till 10 μg signal.

34

Figure 2-9 Dynamic range of milk protein with silver staining

Panel A depicts the original gel image after silver staining and panel B depicts intensity v/s quantity relationship of major milk proteins

It was observed that the lowest detectable milk protein signal was from 50 ng of

total milk protein. On establishing the dynamic range from 50 ng to highest standard, it

was observed that the signal started saturating at the 2.5 μg and linearity deteriorated

35

steadily after that. The dynamic range of the silver stain was thus established from 50 ng

to 2.5 μg. However, it was also observed the time of staining also played a part in

saturating a signal. If on the same gel there are extreme quantities of proteins, the

higher protein quantity signal stained faster and darker and got saturated by the time the

lower quantity protein signal could be adequately stained. Hence, a working dynamic

range for the milk protein standards was established from 100 ng to 1.5 μg, with

acceptable linearity, which was needed for quantification. The R2 for each of the four

bands – BSA, Caseins, β lactoglobulin and α lactalbumin for this dynamic range was

calculated to 0.95, 0.94, 0.97 and 0.96, respectively.

36

Chapter 3. Methodology development for milk adsorption studies

3.1. Optimisation of washing efficiency

To get a reproducible quantification, it is essential to ensure that the protein

constitution does not change during handling. Loosely adsorbed proteins can desorb

non-reproducibly by a slight change in handling or human errors and thus can change

the composition of the adsorbed protein layer affecting accuracy and reproducibility.

Hence, for this project, we will only be investigating tightly adsorbed proteins. To ensure

that only tightly adsorbed proteins are quantified, the vial surface must be thoroughly

washed numerous times to wash off all loosely adsorbed proteins. The experiment is

designed to establish and optimise washing efficiency.

A 100 μL of 1 μg/μL, of milk protein solution in PBS buffer was incubated

overnight in a 0.65 mL polypropylene vial. Following the overnight incubation period, the

supernatant protein solution was discarded and the vial was rinsed with a fresh aliquot of

100 μL PBS with 10x pipetting and the washing was collected in a separate vial. To the

original vial, a fresh aliquot of 100 μL of PBS was introduced and vortexed for 15

seconds and the aliquot was collected. This step was repeated three more times. Finally,

to the vial 100 μL of stripping solution was introduced and incubated in the vial for 15

min. All the washes and strippings were collected, freeze-dried and subjected to gel

electrophoresis and subsequent staining and quantification. Milk proteins with quantities

0.1 µg, 0.3 µg and 1.5 µg was used as standards for quantification (wells 12 to 14).

It was observed that the protein signal is maximum in the first washing and

subsequently diminishes in each of consecutive washings, reaching very low limits after

wash 3 as shown in Figure 3.1, panel A, wells 4, 5 and 10 and hence wash 3 was

arbitrarily chosen as the cut-off point. The protein signal is seen at the stripping sample.

The diminishing protein signal in sequential washes points towards, loosely

adsorbed protein getting washed off in each attempt. The strong signal in the stripping

lane (well 6 and 11) shows that the tightly adsorbed proteins were stripped off from the

surface by the stripping agent. Since the amount of protein recovered in the fourth and

37

fifth washes were insignificant as compared to first three washes, therefore three

washes were sufficient to wash off all the loosely adsorbed proteins from the vial

surface.

Figure 3-1 Optimization of washing efficiency

The figure shows gel image of for washing efficiency. Lanes 1 to 5 and 7 to 10 sequentially shows a reduction in signal intensity, pointing towards diminishing protein quantity in each wash. A spike in well 6 and 11 depicts stripping step to quantify tightly adsorbed proteins

3.2. Optimization of stripping agent

Stripping is a physical separation process, based on the process of mass transfer

that involves removal of one or more components by a stream of liquid or vapour. In my

case stripping can be defined as removal of tightly adsorbed proteins from the vial

38

surface into the stripping solution for quantification. For accurate quantification of protein

adsorption, it is essential that all the adsorbed proteins are stripped off from the vial

surface into the solution for subsequent freeze drying and analysis. An efficiency of

stripping is dependent on solvation property of the reagent and/or its pH.109 Depending

upon the nature of protein adsorbed, the stripping agent must be optimised. A stripping

agent that has very good efficiency for stripping one protein from a vial surface, may not

be equally efficient in another set of protein(s)- vial pair, and hence must be optimised.

Literature reports 0.5% SDS solution as a good stripping agent.16 However, using

this in my protocol gave irreproducible results, which prompted an investigation about

the stripping efficiency of 0.5% SDS solution.

A 100 μL of 1 μg/μL, of milk protein solution in PBS buffer was incubated

overnight in three 0.65 mL polypropylene vials. Following the overnight incubation

period, the vials were washed as described in earlier section. The vials were then

stripped by 100 μL 0.5% SDS solution and the strippings were collected. The vials were

then washed with 100 μL of water to wash off any 0.5% SDS solution remaining. Each of

the vials was then stripped for the second time by 100 μL solution of 1 M HCl, 100 mM

NH4HCO3 and 1 M NH4OH, respectively. Milk proteins with quantity 0.1 µg, 0.5 µg and

2.5 µg were used as standards for quantification (wells 13 to 15).

Protein signal was observed in 0.5% SDS stripping, in wells 1, 5 and 9 as seen in

panel A of Figure 3.2. However, there was also a considerable intense signal observed

in the secondary stripping from HCl, NH4HCO3 and NH4OH (wells 3, 7 and 11). This

proved the inefficiency of 0.5% SDS solution to effectively strip off all the adsorbed milk

proteins from the vial surface. Hence, a new stripping agent had to be optimised for milk

proteins.

39

Figure 3-2 Need for optimisation of new stripping agent

The figure depicts the original gel image, for the failure of 0.5% SDS solution as efficient stripping agent for milk proteins

3.2.1. Organic and inorganic stripping agents

Since, 0.5% SDS solution, could not be used for stripping milk proteins, various

other stripping agents had to be screened for their efficiency. As mentioned earlier,

stripping efficiency is related to solvation and pH, various organic and inorganic reagents

covering pH range from 3 to 11 were screened for their stripping efficiency.

A 100 μL of 1 μg/μL, of milk protein solution in PBS buffer was incubated

overnight in six 0.65 mL polypropylene vials. Following the overnight incubation period,

the vials were washed as described in earlier section. The vials were then stripped by

100 μL of 0.5% SDS solution, 80% ACN, 100 mM HCl, 1 M HCl, 100 mM NH4HCO3, 1 M

NH4OH. All the strippings were collected, freeze-dried and subjected to further SDS-

PAGE analysis. Milk proteins with quantity 0.1 µg, 0.5 µg and 2.5 µg were used as

standards for quantification (wells 13 to 15).

40

The protein signal was highest in 1 M HCl, followed by 100 mM NH4HCO3 and

1 M NH4OH as seen in wells 1 to 6 in panel A of Figure 3.3. However, HCl and NH4OH

are very harsh for the instruments such as Freeze Dryer and hence the third contender,

100 mM NH4HCO3 was selected as a stripping agent.

Figure 3-3 Quantification of various chemicals for stripping efficacy

Figure depicts the original gel image for screening of neat reagents

for stripping efficiency

3.2.2. Selecting SDS-NH4HCO3

SDS is a surfactant that literature reported to be a good stripping agent.10

Though 0.5% SDS solution proved to be an inefficient stripping agent, it's logical to test

SDS efficiency at a higher concentration such as 1% or 2%. Since, stripping solution can

be a mixture of more than one components, mixing equal 100 mM NH4HCO3 with SDS

41

solution may have proved to be a superior stripping agent than a single component

stripping agents.

A 100 μL of 1 μg/μL, of milk protein solution in PBS buffer was incubated

overnight in six 0.65 mL polypropylene vials. Following the overnight incubation period,

the vials were washed as described in earlier section. The vials were then stripped by

100 μL of 0.5% SDS solution, 1% SDS solution, 2% SDS solution, a mixture of

100 mM NH4HCO3-0.5% SDS and a mixture of 100 mM HCl-0.5% SDS sequentially as

shown in panel A of figure 3.4. After using 100 mM NH4HCO3-0.5% SDS and 100 mM

HCl-0.5% SDS as stripping agent, the vials were stripped with 100 mM HCl solution to

assess residual protein signal left by these new stripping agents. All the stripping was

collected, freeze-dried and subjected to further SDS-PAGE analysis. Milk proteins with

quantity 0.1 µg, 0.5 µg and 2.5 µg were used as standards for quantification (wells 13 to

15).

It was observed that Increasing SDS concentration to 1% and 2% did not

significantly improve stripping efficiency (wells 2 and 3). However, 100 mM NH4HCO3-

0.5% SDS mixture, proved to be far more superior than other stripping agents with

regards to stripping efficiency as seen in well 5 and 8. A negligible protein signal was

seen in a tertiary stripped solution of 100 mM HCl as seen in wells 6 and 9 suggesting a

good efficiency of these stripping agents. Hence, for subsequent experiments

100 mM NH4HCO3-0.5% SDS would be used as a primary stripping agent.

42

Figure 3-4 Quantification of various chemical mixtures for stripping efficacy

The figure depicts the original gel for the screening of various chemical mixture as stripping solutions

3.2.3. Reproducibility of 100 mM NH4HCO3-0.5% SDS (ABC)

To incorporate 100 mM NH4HCO3-0.5% SDS (ABC) as a primary stripping agent

is all the experiments, it was essential to prove its efficiency and reproducibility. One way

of validating the reproducibility was to strip the vial first with 100 mM NH4HCO3-0.5%

SDS followed by a secondary and tertiary stripping of the same vial with a harsher

stripping agent.

A 100 μL of 1 μg/μL, protein solution in PBS buffer was incubated overnight in

three 0.65 mL polypropylene vials. Following the overnight incubation period, the vials

were washed as described in earlier section. The vials were then stripped by 100 μL of

100 mM NH4HCO3-0.5% SDS. Each of the vials was then sequentially stripped by

43

100 μL of 80% ACN and 1M HCl. All the stripping was collected, freeze-dried and

subjected to further SDS-PAGE analysis in a sequence shown in panel A of Figure 3.5.

Milk proteins with quantity 0.1 µg, 0.5 µg and 2.5 µg were used as standards for

quantification (wells 13 to 15).

As expected the highest protein signal was obtained from

100 mM NH4HCO3-0.5% SDS (wells 1, 4, 7 and 10) when used as a primary stripping

agent. Secondary and tertiary stripping of the same vial using 80% ACN and 1 M HCl

however, had negligible protein signal. This indicates that 100 mM NH4HCO3-0.5% SDS

could strip off most of the adsorbed proteins from the vial surface.

Figure 3-5 Reproducibility of 100 mM NH4HCO3-0.5% SDS as stripping agent

Figure depicts the original gel image, for validation of reproducibility of new stripping agent

44

3.3. Volume test

To further ensure that the 100 μL of 100 mM NH4HCO3-0.5% SDS is enough to

strip all the proteins from the vial surface and that the stripping agent itself is not getting

saturated by the proteins, thus reducing its efficiency; the surface can be stripped by a

greater volume of stripping agent.

A 100 μL of 1 μg/μL, of milk protein solution in PBS buffer was incubated

overnight in two 0.65 mL polypropylene vials. Following the overnight incubation period,

the vials were washed as described in earlier section. The vials were stripped by 100 μL

of

100 mM NH4HCO3-0.5%SDS. The vials were then rinsed with water to wash off any

residual stripping agent from the previous step. One of the vials were then stripped by

200 μL of 100 mM NH4HCO3-0.5 % SDS and the other vial by 400 μL of 100 mM

NH4HCO3-0.5% SDS. Finally, both the vials were stripped by

1 M HCl as a tertiary stripping agent. All the stripping was collected, freeze-dried and

subjected to further SDS-PAGE analysis in a sequence shown in panel A of Figure 3.6.

Milk proteins with quantity 0.1 µg, 0.3 µg and 1.5 µg were used as standards for

quantification (wells 12 to 14).

Primary stripping of 100 mM NH4HCO3-0.5% SDS displayed maximum protein

signal (wells 1 and 6). There was, however, negligible protein signal when 100 mM

NH4HCO3-0.5% SDS was used as secondary stripping agent at a higher volume (wells 3

and 8) or when 1 M HCl (wells 5 and 10) was used as a tertiary stripping agent. This

validates that 100 μL of 100 mM NH4HCO3-0.5% SDS can effectively strip off all the

adsorbed milk proteins when used as a primary stripping agent, without itself getting

saturated.

45

Figure 3-6 Volume test of 100 mM NH4HCO3-0.5% SDS as stripping agent

Figure depicts the original gel image for volume test experiment for the new stripping agent

3.4. Concentration test

To ensure that 100 mM NH4HCO3-0.5% SDS can strip off all adsorbed proteins

even when a high concentration of milk than used in all the experiments for this project,

is used. The stripping agent must be tested for its effectiveness at higher milk

concentration.

A 100 μL of 5 μg/μL, of milk protein solution in PBS buffer was incubated

overnight in two 0.65 mL polypropylene vials. Following the overnight incubation period,

the vials were washed as described in earlier section. The vials were then stripped by

100 μL of 100 mM NH4HCO3-0.5% SDS. The vials were then rinsed with water to wash

off any residual stripping agent from the previous step. Both the vials were then stripped

by 100 μL of 100 mM NH4HCO3-0.5% SDS. The vials were again rinsed with water to

wash off residual stripping solution from the earlier step. Both the vials were stripped by

80% ACN as tertiary stripping agent, followed by quaternary stripping by 1 M HCl. All

the stripping was collected, freeze-dried and subjected to further SDS-PAGE analysis in

46

a sequence shown in panel A of figure 3.7. Milk proteins with quantity 0.1 µg, 0.3 µg and

1.5 µg were used as standards for quantification (wells 12 to 14).

As expected highest protein signal came from 100 mM NH4HCO3-0.5% SDS

when used as a primary stripping agent (wells 1 and 7). There was no detectable signal

from water washes or the 100 mM NH4HCO3-0.5% SDS as a secondary stripping agent

(wells 3 and 9). There was negligible protein signal from tertiary and quaternary stripping

agent, but the signal is insignificant when compared to primary stripping agent (wells 6

and 11). The experiment proves that 100 mM NH4HCO3-0.5% SDS can strip milk

proteins off the vial surface even when the concentration of incubated milk protein is

many folds higher than that used in other experiments. This further bolsters the

reproducibility and an efficacy of 100 mM NH4HCO3-0.5% SDS as a good stripping

agent.

Figure 3-7 Concentration test of 100 mM NH4HCO3-0.5% SDS as stripping agent

47

Figure depicts the original gel image for concentration test experiment for the new stripping agent

In conclusion, we were successful in designing a new stripping agent that was

more efficient than 0.5% SDS solution in scrapping off tightly adsorbed milk proteins

from various surfaces. The agent was tested for its reproducibility and robustness was

proved to be equally efficient for various surfaces. The near neutral pH of the agent also

amicable for use with expensive instruments.

48

Chapter 4. Studies on milk protein adsorption

4.1. Concentration saturation

To correctly quantify the adsorbed proteins, it is essential to ensure that all the

available surface is completely saturated with adsorbed proteins. This can be achieved

by incubating the surface vials with increasing concentration of protein solutions and

then quantifying the adsorbed protein quantity for each concentration.

For this experiment, 0.65 mL polypropylene vials were incubated overnight with

100 μL of 1 ng/μL, 5 ng/μL, 50 ng/μL, 500 ng/μL, 1 μg/μL, 5 μg/μL and 7 μg/μL of milk

proteins in PBS buffer. After the above-described incubation period, the vials were

washed and stripped per earlier described procedure and subjected to freeze drying and

gel electrophoresis, freeze-dried and subjected to further SDS-PAGE analysis in a

sequence shown in panel A of Figure 4.1. Milk proteins with quantity 0.1 µg, 0.3 µg and

1.0 µg were used as standards for quantification of whey proteins (wells 7, 6 and 5

respectively). The second set of commercially available pure β casein standards with

quantity 0.1 µg, 0.3 µg and 1.0 µg was also incorporated for quantification of β casein

(well 2 to 4).

Due to the limitation on a number of wells in a gel, two gels were used for this

experiment (Figure 4.1, panels A1 and A2). Each gel had its own standard curve for

quantitation. (panels B and C). A standard curve for β casein was obtained from lanes 2,

3 and 4 (Panel C2) while wells 5, 6 and 7 provided standard curve for other proteins

(Panel C1) A combined plot of total milk proteins adsorption obtained from both the gels

was also plotted to observe surface saturation (panel D).

A revealed that the quantity of adsorbed proteins increased with the rise in

concentration, and attained stagnation at concentration 50 ng/μL, after which the

quantity adsorbed was very similar to other greater concentrations (wells 12 and 13).

There was, however, a noticeable increase in adsorbed protein quantity at 1 μg/μL (wells

8 and 9). This increase can be explained as the change in protein orientation from end-

49

on to side-on once the critical surface protein density is attained.8 The literature reported

value for the critical concentration of whey and caseins in milk is 200 ng/μL.8,110 Since, in

this experiment, there were no data points around this value, the change in orientation

was observed at next data point, 1 μg/μL Thus, it can be concluded that all available

surfaces were saturated at a concentration of 50 ng/μL, and hence, this was the

optimized protein concentration that was used in subsequent experiments.

50

Figure 4-1 Concentration saturation of polypropylene vial surfaces at

various protein concentrations

Panel A depicts the original gel image, while panel B shows graphical representation for concentration saturation of individual and total milk proteins. Panel C1 and C2 are the standard curve used for quantitation for β casein and other proteins respective

4.2. Time saturation

Protein adsorption is a time dependent process. Hence, even after optimising the

concentration required to surface saturation, the time required for the proteins to occupy

all the available surface should be ascertained as well. This can be achieved by

incubating the surface vials with a same concentration of 50 ng/μL, but for increasing the

51

time of incubation, followed quantification of the adsorbed protein quantity for each time

point.

For this experiment, 0.65 mL polypropylene vials were incubated with 100 μL of

50 ng/μL, of milk proteins solution in PBS buffer for 3 hrs, 5 hrs, 8 hrs, 12 hrs and 16 hrs.

Following this, the vials were washed and stripped per earlier described procedure and

subjected to freeze drying and gel electrophoresis analysis in a sequence shown in

panel A of Figure 4.2. Milk proteins with quantity 0.1 µg, 0.3 µg and 1.0 µg were used as

standards for quantification of whey proteins (wells 7, 6 and 5 respectively). The second

set of commercially available pure β casein standards with quantity 0.1 µg, 0.3 µg and

1.0 µg was also incorporated for quantification of β casein (well 2 to 4).

In this experiment, due to the low background, an intense α casein band was

observed, which was easily quantifiable. Hence, in this experiment, all five major protein

bands have been quantified. Data analysis revealed that the quantity of adsorbed

proteins increased with increase in time, and attained stagnation at after 8 hrs, after

which the quantity adsorbed was very like other greater time points, with no changes in

the internal ratios of protein bands (wells 6 to 11). The initial steady rise in protein

quantity adsorbed appears gives rise to uneven variation as time passes (panel B). As

the time passes it gives proteins time to increase their footprint on the surface.8 During

this processes protein that has higher affinity to the surface, may try to dislodge proteins

with lower surface affinity, to increase their footprint. This competition continues till an

equilibrium state is achieved.8,17,111 The time required for this process is roughly 8 hours

for milk proteins. Thus, it can be concluded that all available surfaces were saturated at

8 hrs, and hence, this was the optimized incubation time that was used in subsequent

experiments.4,112

52

53

Figure 4-2 Time saturation of polypropylene vial surfaces at various incubation periods

Panel A depicts the original gel image, while panel B shows graphical representation for time saturation of individual milk and total milk proteins. Panel C1 and C2 are the standard curve used for quantitation for β casein and other proteins respectively

54

4.3. Effect of temperature

As discussed in the earlier chapter, temperature influences kinetics and

thermodynamics of protein adsorption and thus is one of the important extrinsic factors

affecting adsorption. At higher temperature, micelle proteins such as caseins lose water

molecules or ions trapped in their micelles, which essentially disrupts the micelle,

making it prone to adsorption.67 Also, at a higher temperature, owing to increased

diffusion of proteins towards the surface, may also contribute to more non-specific

protein adsorption. Thus, quantifying the effect of temperature on milk proteins would

give useful data on the behaviour of different proteins towards temperature.

For this experiment, 8 vials were incubated with 100 μL of 50 ng/μL of milk

protein solution in PBS buffer was incubated overnight. The incubation temperature was

however changed for an each pair. Each set of vials was incubated at -20 °C, 4 °C, 20 °C

(R.T.) and 37 °C. Following the lapse of the incubation period, the vials were washed,

stripped and subjected to gel electrophoresis as described in earlier section. Milk

proteins with quantity 0.1 µg, 0.3 µg and 1.0 µg were used as standards for

quantification of whey proteins (wells 7, 6, and 5 respectively). The second set of

commercially available pure β casein standards with quantity 0.1 µg, 0.3 µg and 1.0 µg

was also incorporated for quantification of β casein (well 2 to 4). During sample

incubation step, care was taken to minimize subjecting samples to changing

temperature. All the solvents, vials and pipette tips, were cooled to at -20 °C or 4 °C

before use. Since all the solvents and protein solutions were frozen at -20 °C, the signal

at -20 °C shows the absorption that took place while the solution was freezing and

thawing.

Analysis revealed that the general trend increased in adsorbed protein quantity

with the increase in temperature.8,37,113 This is due to two major reasons, the first being

an increase in protein kinetics with the rise in temperature and second an increase

protein adsorption caused by loss of core water molecules or ions due to increased

temperature.8,111 However, caseins and β lactoglobulin were found to be more sensitive

to the change in temperature as compared to BSA and α lactalbumin. It was also

observed that there was a preference for caseins for adsorption as compared to whey

proteins.

55

Figure 4-3 Analysis of effect of temperature on milk protein adsorption

on polypropylene

Figure represents effect of temperature on individual milk proteins Note- The image was converted to black and white (8 bit) in

ImageJ for quantification to cancel out background

4.4. Effect of pH

Isoelectric points of protein macromolecules make them susceptible to changes

in pH. The degree of deviation of solution’s pH from the isoelectric point of proteins can

impart a positive, negative or no charge on the proteins, which can completely alter the

adsorption pattern of proteins. Thus, to study the effect of pH on milk protein adsorption,

it is essential to subject milk proteins to different pH and quantify the adsorbed proteins.

56

For this experiment, pH of regular PBS solution was altered using strong acids or

alkalis and PBS with pH 3, 5, 7, 9 and 11 were prepared. The altered pH of PBS was

confirmed by pH paper and pH meter. For this experiment, 10 vials were incubated with

100 μL of 50 ng/μL of milk protein solution in PBS buffer (altered pH) were incubated

overnight at room temperature. At the sample preparation step, the protein solution was

diluted from the stock using one of the above pHs. Following this, the milk solutions were

incubated, washed, stripped and subjected to the gel electrophoresis as described

earlier. Milk proteins with quantity 0.1 µg, 0.3 µg and 1.0 µg were used as standards for

quantification of whey proteins (wells 7, 6 and 5 respectively). The second set of

commercially available pure β casein standards with quantity 0.1 µg, 0.3 µg and 1.0 µg

was also incorporated for quantification of β casein (well 2 to 4).

Analysis showed a marked increase in adsorbed protein quantity at pH 5. Since

the isoelectric point of most of the milk proteins is around pH 5, the net charge on them

is zero, which facilitates dense packing of protein molecules during adsorption, leading

to increased adsorption.8,37,114 The general trend was that the effect of pH on adsorption

decreases with increase in pH. Adsorbed protein quantities were comparable for pH 3

and 7, but decreased at pH 9 and were minimum at pH 11. Owing to the micelle

structure, caseins were observed to be more sensitive to changes in pH than whey

proteins.37,60,115

57

Figure 4-4 Quantification of the effect of pH on milk protein adsorption on polypropylene vials (Gel 1)

Figure depicts the original gel image of the effect of pH on

individual milk proteins

Following is the quantitation of gel two that had samples for pH 7 and pH 11

along with milk and β casein standards

58

Figure 4-5 Quantification of effect of pH on milk protein adsorption on polypropylene vials (Gel 2)

Figure depicts the original gel image of the effect of pH on individual

milk proteins

4.5. Effect of surfaces

Nature of adsorbent surface is a critical component influencing protein

adsorption. Adsorbent related factors such as hydrophobicity and surface charges in

relation to the nature of proteins can change the adsorption spectrum of the proteome.

Proteins tend to adsorb preferably to hydrophobic surfaces. Further, adsorption

increases if the protein and surface are oppositely charged. Thus, quantifying the effect

of different surfaces on milk proteins provides an opportunity to study this influence.

For this experiment, 12 vials were incubated with 100 μL of 50 ng/μL of milk

protein solution in PBS buffer was incubated overnight at room temperature. Protein

solutions were incubated in four different surface vials – glass, uncoated polypropylene

vial, coated polypropylene vial and low binding mass spectrometry (LBMS) grade vial.

Care was taken to subject protein solutions to the same surface area so as not to

change adsorption spectrum. Following, incubation, the vials were washed, stripped and

subjected to gel electrophoresis as described earlier.

59

It was observed that the adsorption was more on uncoated vials than coated

vials. In agreement with the literature, protein adsorption was found to be maximum on

glass followed by uncoated polypropylene vial and least on coated polypropylene vial

and LBMS vials.12,116 In my study, however, in contradiction to the literature reports on

the preference of β casein for adsorption, it was observed that whey proteins were more

sensitive to protein-surface interactions as compared to caseins.54,79,117 An important

observation from this experiment was that, even though the coated polypropylene and

LBMS vials are marketed as adsorption resistant, they still showed a detectable quantity

of proteins. This observation validates the problem of non-specific protein adsorption

occurring on these vials.

Figure 4-6 Analysis of the effect of nature of adsorbate surfaces on milk

protein adsorption on polypropylene vials

Panel A depicts the original gel image for effect of changes in nature of surfaces on individual milk proteins. Note- The image was converted to black and white (8 bit) in ImgaeJ for quantification to

cancel out background

60

Chapter 5. Conclusion and future work

5.1. Conclusion

A simple and direct protein quantification method (SDS-PAGE) that was

developed in our lab was further improved to ease quantification of complex protein

mixtures. Various organics, inorganic and detergents were screened as potential

stripping agents. A completely new stripping agent was developed that is capable of

stripping off proteins varying in their physical and chemical properties off polypropylene

and glass surfaces. The agent was thoroughly tested for its efficiency, sensitivity and

reproducibility and the 100 mM NH4HCO3-0.5% SDS proved its mettle in all the tests.

This stripping agent was then used to quantify skim milk protein adsorption

spectrum under various conditions namely temperature, pH and surfaces to study

differential adsorption spectrum of the proteome. The data obtained from studying these

three factors has paved way to incorporate additional factors to study differential

adsorption spectrum. Various permutation-combination operations on the data thus

obtained can be used to narrow down on factors that could be manipulated to minimise

non-specific protein adsorption depending upon the physiochemical properties of the

involved proteins. The study also helped to observe competitive adsorption behaviour

among proteins, which can potentially have exploited to prevent unwanted loss of low

abundant proteins. The information gathered has provided a preliminary direction to

manage non-specific protein adsorption in samples.

5.2. Future work

There is an immense desire from pharmaceutical industry to reduce non-specific

protein adsorption for bioactive large molecules such as vaccines, sera and toxoids.

With the introduction of protein based drugs such as antibodies and antivenins as

therapeutic agents has opened a lucrative but challenging market. Due to the peptide

61

nature of these drugs they have potential to be non-specifically adsorbed from

production to shipping. Much of proteins’ characteristics depend upon their internal

molecular make up and their interactions with the external surrounding environment.118

This series of events decide the secondary and tertiary nature of proteins. During

adsorption, proteins lose their native state which may affect its efficacy. Apart from

pharmaceutical industry, biomedical engineers and analytical chemists struggle with

nonspecific protein adsorption as it routinely leads to problems ranging from implant

rejection to saturation of analytical detectors.8 Dairy industry has also been struggling to

find ways to arrest NSPA phenomenon that significantly reduces life time of their

equipments.121

A future work of this project would be to increase the number of external and

internal factors such as ion concentration in protein solution, surface energy, polarity,

charges and morphology of surfaces and proteins. Using mass spectrometry based

proteomic techniques it is possible to elucidate the structure of individual proteins even

in most complex mixtures. Since individual proteins are unique in their physiochemical

properties, it is possible to reduce their adsorption by tailoring the external factors that

reduce adsorption under those conditions. Once this information is gathered for milk

proteins, the same exercise can be performed for more complex proteomes such as cell

lysates and also by testing protein adsorption on other surfaces. The knowledge gained

from these experiments will help us to design strategies to reduce non-specific protein

adsorption.

Quantification of adsorbed proteins is another avenue that deserves more

investigation. As mentioned in earlier chapters, we used silver staining for protein

quantification because of its high sensitivity. However, the problem of low linearity and

rapid signal saturation remains a major blockade along with other issues such as

staining bias and appearance of additional β casein bands. Though we tried various

methods such as alkylating the proteins to prevent the appearance of additional band,

the problem still remains unsolved. Improving the silver stain reproducibility or exploring

other avenues of staining or quantification of proteins will be an important future work.

62

References

1. Dabrowski, A. Adsorption - From theory to practice. Adv. Colloid Interface Sci. 93, 135–224 (2001).

2. Merriam-Webster.com. ‘Adsorbent’. Merriam-Webster.com at <https://www.merriam-webster.com/dictionary/adsorbent>

3. Merriam-Webster.com. ‘adsorbate’. Merriam-Webster.com (2017). at <Merriam-Webster.com>

4. Norde, W. & Anusiem, A. C. I. Adsorption, desorption and re-adsorption of proteins on solid surfaces. Colloids and Surfaces 66, 73–80 (1992).

5. Andrade, J. D., Hlady, V. & Wei, a. P. Adsorption of complex proteins at interfaces. Pure and Applied Chemistry 64, 1777–1781 (1992).

6. Chen, J. & Dickinson, E. Protein/surfactant interfacial interactions Part 3. Competitive adsorption of protein+surfactant in emulsions. Colloids Surfaces, A

Physicochem. Eng. Asp. 101, 77–85 (1995).

7. Hlady, V. & Buijs, J. Protein adsorption on solid surfaces. Curr. Opin. Biotechnol. 7, 72–7 (1996).

8. Rabe, M., Verdes, D. & Seeger, S. Understanding protein adsorption phenomena

at solid surfaces. Adv. Colloid Interface Sci. 162, 87–106 (2011).

9. Vogler, E. A. Protein adsorption in three dimensions. Biomaterials 33, 1201–1237 (2012).

10. Lee, M. C. G., Wu, K. S. Y., Nguyen, T. N. T. & Sun, B. Sodium dodecyl sulfate polyacrylamide gel electrophoresis for direct quantitation of protein adsorption. Anal. Biochem. 465, 102–104 (2014).

11. Shimizu, M., Kamiya, T. & Yamauchi, K. The adsorption of whey proteins on the surface of emulsified fat. Agric. Biol. Chem. 45, 2491–2496 (1981).

12. Varmette, E., Strony, B., Haines, D. & Redkar, R. An assay for measurement of protein adsorption to glass vials. PDA J. Pharm. Sci. Technol. 64, 305–15 (2010).

13. Suelter, C. H. & DeLuca, M. How to prevent losses of protein by adsorption to glass and plastic. Anal. Biochem. 135, 112–119 (1983).

14. Norde, W. Protein adsorption at solid surfaces: A thermodynamic approach. Pure Appl. Chem. 66, 491–496 (1994).

15. Su, T., Lu, J., Thomas, R., Cui, Z. & Penfold, J. The Adsorption of Lysozyme at

63

the Silica-Water Interface: A Neutron Reflection Study. J. Colloid Interface Sci. 203, 419–29 (1998).

16. Gilbert, L. & Lee Gilbert. Method Development for an Easy and Direct Quantitation of Protein Adsorption by Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE). (2014).

17. Cuypers, P. A., Willems, G. M., Hemker, H. C. & Hermens, W. T. Adsorption kinetics of protein mixtures. A tentative explanation of the Vroman effect. Ann. N.

Y. Acad. Sci. 516, 244–252 (1987).

18. Grygorczyk, B. A. Biophysical Studies on Milk Protein Interactions in Relation to Storage. (2009).

19. Caruso, F. et al. Adsorption dynamics of surfactants at the air/water interface: a critical review of mathematical models, data, and mechanisms. J. Chem. Inf. Model. 53, 1689–1699 (2013).

20. Gettens, R. T. T., Bai, Z. & Gilbert, J. L. Quantification of the kinetics and thermodynamics of protein adsorption using atomic force microscopy. J. Biomed.

Mater. Res. A 72, 246–257 (2005).

21. Krisdhasima, V., Vinaraphong, P. & McGuire, J. Adsorption Kinetics and Elutability of α-Lactalbumin, β-Casein, β-Lactoglobulin, and Bovine Serum Albumin at Hydrophobic and Hydrophilic Interfaces. J. Colloid Interface Sci. 161,

325–334 (1993).

22. Wilkins, M. R. et al. From Proteins to Proteomes: Large Scale Protein Identification by Two-Dimensional Electrophoresis and Arnino Acid Analysis. Nat Biotech 14, 61–65 (1996).

23. Binder, H. et al. Time-course human urine proteomics in space-flight simulation experiments. BMC Genomics 15, S2 (2014).

24. Anderson, N. L. & Anderson, N. G. Proteome and proteomics: new technologies, new concepts, and new words. Electrophoresis 19, 1853–1861 (1998).

25. Liu, T. et al. Evaluation of multiprotein immunoaffinity subtraction for plasma proteomics and candidate biomarker discovery using mass spectrometry. Mol. Cell. Proteomics 5, 2167–2174 (2006).

26. Kim, J. Protein adsorption on polymer particles. J. Biomed. Mater. Res. 21, 4373–

4381 (2002).

27. Charles, P. T. et al. Reduction of non-specific protein adsorption using poly(ethylene) glycol (PEG) modified polyacrylate hydrogels in immunoassays for staphylococcal enterotoxin B detection. Sensors 9, 645–655 (2009).

64

28. Wahlgren, M. & Arnebrant, T. Protein adsorption to solid surfaces. Trends Biotechnol. 9, 201–208 (1991).

29. Cornec, M. & Narsimhan, G. Effect of contaminant on adsorption of whey proteins

at the air-water interface. J. Agric. Food Chem. 46, 2490–2498 (1998).

30. Elwing, H., Askendal, A. & Lundström, I. Competition between adsorbed fibrinogen and high-molecular-weight kininogen on solid surfaces incubated in human plasma (the vroman effect): Influence of solid surface wettability. J.

Biomed. Mater. Res. 21, 1023–1028 (1987).

31. Lee, J. H., Kopecek, J. & Andrade, J. D. Protein‐resistant surfaces prepared by PEO‐containing block copolymer surfactants. J. Biomed. Mater. Res. 23, 351–368

(1989).

32. Cooper, A. Conformational fluctuation and change in biological macromolecules. Sci. Prog. 66, 473 (1980).

33. Latour, R. a. & Rini, C. J. Theoretical analysis of adsorption thermodynamics for hydrophobic peptide residues on SAM surfaces of varying functionality. J. Biomed. Mater. Res. 60, 564–577 (2002).

34. Ha, J.-H., Spolar, R. S. & Record, M. T. Role of the hydrophobic effect in stability

of site-specific protein-DNA complexes. J. Mol. Biol. 209, 801–816 (1989).

35. MIRIANI, M. Protein unfolding on interfaces : a structural and functional study. (Universita Degli Studi Di Milano, 2011).

36. Malmsten, M. Formation of Adsorbed Protein Layers. J. Colloid Interface Sci. 207,

186–199 (1998).

37. Bingham, E. W. Influence of temperature and pH on the solubility of αs1-, β- and κ-casein. J. Dairy Sci. 54, 1077–1080 (1971).

38. McKenzie, Hugh (Department of Physical Biochemistry, Institute of Advanced Studies, Australian National University, Canberra, A. Milk Proteins - Chemistry and Molecular Biology. (Academic Press, 1970).

39. Whitney, R. M. et al. Nomemclature of the proteins of cow’s milk: fourth revision. J. Dairy Sci. 59, 795–815 (1976).

40. Swaisgood, H. E. Review and update of casein chemistry. J. Dairy Sci. 76, 3054–3061 (1993).

41. Phadungath, C. Casein micelle structure: a concise review. Songklanakarin J. Sci. Technol. 27, 201–212 (2005).

42. Rabe, M., Verdes, D., Rankl, M., Artus, G. R. J. & Seeger, S. A comprehensive

65

study of concepts and phenomena of the nonspecific adsorption of β-lactoglobulin. ChemPhysChem 8, 862–872 (2007).

43. Wu, C. S. & Chen, G. C. Adsorption of proteins onto glass surfaces and its effect on the intensity of circular dichroism spectra. Anal. Biochem. 177, 178–182 (1989).

44. Krishnan, A., Liu, Y.-H., Cha, P., Allara, D. & Vogler, E. A. Interfacial energetics of globular-blood protein adsorption to a hydrophobic interface from aqueous-buffer

solution. J. R. Soc. Interface 3, 283–301 (2006).

45. Wu, C.-S. C. & Chen, G. C. Adsorption of proteins onto glass surfaces and its effect on the intensity of circular dichroism spectra. Anal. Biochem. 177, 178–182 (1989).

46. Nylander, T., Kékicheff, P. & Ninham, B. W. The Effect of Solution Behavior of Insulin on Interactions between Adsorbed Layers of Insulin. J. Colloid Interface Sci. 164, 136–150 (1994).

47. Evers, F., Steitz, R., Tolan, M. & Czeslik, C. Analysis of hofmeister effects on the density profile of protein adsorbates: A neutron reflectivity study. J. Phys. Chem. B 113, 8462–8465 (2009).

48. Anand, G., Sharma, S., Dutta, A. K., Kumar, S. K. & Belfort, G. Conformational Transitions of Adsorbed Proteins on Surfaces of Varying Polarity. Langmuir 26,

10803–10811 (2010).

49. Cacace, M. G., Landau, E. M. & Ramsden, J. J. The Hofmeister series: salt and solvent effects on interfacial phenomena. Q. Rev. Biophys. 30, 241–277 (1997).

50. Dickinson, E. Properties of emulsions stabilized with milk proteins: overview of

some recent developments. J. Dairy Sci. 80, 2607–2619 (1997).

51. Dickinson, E. & Eliot, C. Aggregated Casein Gels : Interactions , Rheology and Microstructure. Proceeding 3rd Int. Symp. food Rheol. Struct. 37–44 (2003).

52. Alberts B, Johnson A, Lewis J, et al. Molecular Biology of the Cell. (Garland

Sciences, New York, 2002). at <https://www.ncbi.nlm.nih.gov/books/NBK21054/>

53. Tooze, carl branden and john. Introduction to Protein Structure. (Garland Sciences, New York, 1999).

54. Jr, D. V. N. & Nicolau, D. V. Towards a Theory of Protein Adsorption : Predicting the Adsorption of Proteins on Surfaces Using a Piecewise Linear Model Validated Using the Biomolecular Adsorption Database. Reproduction 1–6 (2004).

55. Norde, W. & Anusiem, A. C. I. Adsorption, desorption and readsorption of proteins on solid-surfaces. Colloids And Surfaces 66, 73–80 (1992).

66

56. Sharpe, J. R., Sammons, R. L. & Marquis, P. M. Effect of pH on protein adsorption to hydroxyapatite and tricalcium phosphate ceramics. Biomaterials 18, 471–476 (1997).

57. Williams, A. & Prins, A. Comparison of the dilational behavior of adsorbed milk proteins at the air-water and oil-water interfaces. Colloids Surfaces, A Physicochem. Eng. Asp. 114, 267–275 (1996).

58. Liu, Y. Some consideration on the Langmuir isotherm equation. Colloids Surfaces

A Physicochem. Eng. Asp. 274, 34–36 (2006).

59. Pankow, J. F. Review and comparative analysis of the theories on partitioning between the gas and aerosol particulate phases in the atmosphere. Atmos. Environ. 21, 2275–2283 (1987).

60. Zhang, Z., Dalgleish, D. . & Goff, H. . Effect of pH and ionic strength on competitive protein adsorption to air/water interfaces in aqueous foams made with mixed milk proteins. Colloids Surfaces B Biointerfaces 34, 113–121 (2004).

61. Dupont-Gillain, C. C., Fauroux, C. M. J., Gardner, D. C. J. & Leggett, G. J. Use of AFM to probe the adsorption strength and time-dependent changes of albumin on self-assembled monolayers. J. Biomed. Mater. Res. A 67, 548–558 (2003).

62. D’Alessandro, A., Zolla, L. & Scaloni, A. The bovine milk proteome: cherishing, nourishing and fostering molecular complexity. An interactomics and functional

overview. Mol. Biosyst. 7, 579 (2011).

63. Sharma, R. & Singh, H. Adsorption behavior of commercial milk protein and milk powder products in low-fat emulsions. Milchwissenschaft 53, 373–377 (1998).

64. Dalgleish, D. G. Casein Micelles as Colloids: Surface Structures and Stabilities. J.

Dairy Sci. 81, 3013–3018 (2017).

65. Chobert, J. M., Bertrand-Harb, C. & Nicolas, M. G. Solubility and emulsifying properties of caseins and whey proteins modified enzymically by trypsin. J. Agric. Food Chem. 36, 883–892 (1988).

66. Mavropoulou, I. P., Kosikowski, F. V. & Moore, S. Composition, Solubility, and Stability of Whey Powders. J. Dairy Sci. 56, 1128–1134 (1973).

67. Post, A. E., Arnold, B., Weiss, J. & Hinrichs, J. Effect of temperature and pH on the solubility of caseins: Environmental influences on the dissociation of alpha(S)-

and beta-casein. J. Dairy Sci. 95, 1603–1616 (2012).

68. De Kruif, C. G., Huppertz, T., Urban, V. S. & Petukhov, A. V. Casein micelles and their internal structure. Adv. Colloid Interface Sci. 171–172, 36–52 (2012).

69. Walstra, P., Geurts, T. J., Noomen, A., Jellema, A. & van Boekel, M. A. J. S. Dairy

67

Technology: Principles of Milk Properties and Processes. Taylor&Francis e Library (2005). doi:10.1111/j.1471-0307.2005.00175.x

70. Wong, N. P., Jenness, R., Keeney, M. & Marth, E. H. Fundamentals of Dairy

Chemistry. Fundam. dairy Chem. 767 (1999).

71. Kinsella, J.E. and Whitehead, D. . in Adv. Food Nutr. Res. 343–438 (1989).

72. Luhovyy, B. L., Akhavan, T. & Anderson, G. H. Whey Proteins in the Regulation of Food Intake and Satiety. J. Am. Coll. Nutr. 26, 704S–712S (2007).

73. Parris, N., Purcell, J. M. & Ptashkin, S. M. Thermal denaturation of whey proteins in skim milk. J. Agric. Food Chem. 39, 2167–2170 (1991).

74. Uhrínová, S. et al. Structural Changes Accompanying pH-Induced Dissociation of the β-Lactoglobulin Dimer † , ‡. Biochemistry 39, 3565–3574 (2000).

75. Bramaud, C., Aimar, P. & Daufin, G. Whey protein fractionation: Isoelectric precipitation of α-lactalbumin under gentle heat treatment. Biotechnol. Bioeng. 56, 391–397 (1997).

76. Bu, Z., Cook, J. & Callaway, D. J. . Dynamic regimes and correlated structural dynamics in native and denatured alpha-lactalbumin. J. Mol. Biol. 312, 865–873 (2001).

77. Reimhult, K., Petersson, K. & Krozer, A. QCM-D analysis of the performance of blocking agents on gold and polystyrene surfaces. Langmuir 24, 8695–8700

(2008).

78. Benesch, J., Askendal, A. & Tengvall, P. Quantification of adsorbed human serum albumin at solid interfaces: A comparison between radioimmunoassay (RIA) and simple null ellipsometry. Colloids Surfaces B Biointerfaces 18, 71–81 (2000).

79. Brouette, N. et al. A neutron reflection study of adsorbed deuterated myoglobin layers on hydrophobic surfaces. J. Colloid Interface Sci. 390, 114–120 (2013).

80. Rojas, E., Gallego, M. & Reviakine, I. Effect of sample heterogeneity on the interpretation of quartz crystal microbalance data: Impurity effects. Anal. Chem.

80, 8982–8990 (2008).

81. Verzola, B., Gelfi, C. & Righetti, P. G. Protein adsorption to the bare silica wall in capillary electrophoresis - Quantitative study on the chemical composition of the background electrolyte for minimising the phenomenon. J. Chromatogr. A 868,

85–99 (2000).

82. Holmberg, M. & Hou, X. Competitive protein adsorption of albumin and immunoglobulin G from human serum onto polymer surfaces. Langmuir 26, 938–942 (2010).

68

83. Leibner, E. S. et al. Superhydrophobic effect on the adsorption of human serum albumin. Acta Biomater. 5, 1389–1398 (2009).

84. Ronald Woodbury & Woodbury, R. High Throughpuut Methods for ‘Deep Proteome’ Analyses for Biomarker Discovery through Equalizer Bead Fractionation an the ProteinChip SELDI System. (HUPO 2003).

85. Ng-Kwai-Hang, K. F. & Kroeker, E. M. Rapid Separation and Quantification of Major Caseins and Whey Proteins of Bovine Milk by Polyacrylamide Gel

Electrophoresis. J. Dairy Sci. 67, 3052–3056 (1984).

86. Weber, K. & Osborn, M. The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J. Biol. Chem. 244, 4406–12 (1969).

87. Schagger, H. & von Jagow, G. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem 166, 368–379 (1987).

88. Raymond, S. & Weintraub, L. Acrylamide gel as a supporting medium for zone

electrophoresis. Science (80-. ). 130, 711 (1959).

89. Burgess2, A. C. G. and R. R. & —. Preparation of protein samples for SDS-polyacrylamide gel electrophoresis: procedures and tips. Innovations 10 (2013). at <http://wolfson.huji.ac.il/purification/PDF/PAGE_SDS/NOVAGEN_Prepare_Sampl

e_PAGE_SDS.pdf>

90. Dyballa, N. & Metzger, S. Fast and sensitive colloidal coomassie G-250 staining for proteins in polyacrylamide gels. J. Vis. Exp. 2–5 (2009). doi:10.3791/1431

91. Georgiou, C. D., Grintzalis, K., Zervoudakis, G. & Papapostolou, I. Mechanism of Coomassie brilliant blue G-250 binding to proteins: A hydrophobic assay for nanogram quantities of proteins. Anal. Bioanal. Chem. 391, 391–403 (2008).

92. Smejkal, G. B. The Coomassie chronicles: past, present and future perspectives in polyacrylamide gel staining. Expert Rev. Proteomics 1, 381–387 (2014).

93. Merril, C. R., Bisher, M. E., Harrington, M. & Steven, a C. Coloration of silver-stained protein bands in polyacrylamide gels is caused by light scattering from silver grains of characteristic sizes. Proc. Natl. Acad. Sci. U. S. A. 85, 453–7 (1988).

94. Rabilloud, T., Vuillard, L., Gilly, C. & Lawrence, J.-J. Silver-staining of proteins in polyacrylamide gels: a general overview. Cell. Mol. Biol. (Noisy-le-grand). 40, 57–75 (1994).

95. Gromova, I. & Celis, J. E. Protein Detection in Gels by Silver Staining: A Procedure Compatible with Mass Spectrometry. Cell Biol. Four-Volume Set 4,

69

219–223 (2006).

96. Wray, W., Boulikas, T., Wray, V. P. & Hancock, R. Silver staining of proteins in polyacrylamide gels. Anal. Biochem. 118, 197–203 (1981).

97. Rabilloud, T. Mechanisms of protein silver staining in polyacrylamide gels: a 10-year synthesis. Electrophoresis 11, 785–794 (1990).

98. Samuel, E. P. The mechanism of silver staining. J. Anat. 87, 278–287 (1953).

99. Winkler, C., Denker, K., Wortelkamp, S. & Sickmann, A. Silver- and Coomassie-staining protocols: Detection limits and compatibility with ESI MS. Electrophoresis 28, 2095–2099 (2007).

100. Leize-wagner, E. & Rabilloud, T. About the Mechanism of Interference of Silver Staining. 1–18

101. Albright, J. C., Dassenko, D. J., Mohamed, E. A. & Beussman, D. J. Identifying gel-separated proteins using in-gel digestion, mass spectrometry, and database searching: Consider the chemistry. Biochem. Mol. Biol. Educ. 37, 49–55 (2009).

102. Gundry, R. L. et al. Preparation of proteins and peptides for mass spectrometry analysis in a bottom-up proteomics workflow. Curr. Protoc. Mol. Biol. (2009). doi:10.1002/0471142727.mb1025s88

103. Darling, D. F. & Butcher, D. W. Quantification of polyacrylamide gel electrophoresis for analysis of whey proteins. Journal of dairy science 59, 863–7

(1976).

104. Nakanishi, K., Sakiyama, T. & Imamura, K. On the adsorption of proteins on solid surfaces, a common but very complicated phenomenon. J. Biosci. Bioeng. 91, 233–244 (2001).

105. Perez, A. A., Sánchez, C. C., Rodríguez Patino, J. M., Rubiolo, A. C. & Santiago, L. G. Surface adsorption behaviour of milk whey protein and pectin mixtures under conditions of air-water interface saturation. Colloids Surfaces B Biointerfaces 85, 306–315 (2011).

106. Yang, Y. et al. Animal species milk identification by comparison of two-dimensional gel map profile and mass spectrometry approach. Int. Dairy J. 35, 15–20 (2014).

107. ter Beek, L. C. et al. Nuclear magnetic resonance study of the conformation and dynamics of beta-casein at the oil/water interface in emulsions. Biophys. J. 70, 2396–2402 (1996).

108. Dickinson, E. Structure of adsorbed protein layers at fluid interfaces. in COLL-020 (American Chemical Society, 1995).

70

109. Abcam. Stripping for reprobing. ABCAM Tech. 1–2 (2013). at <http://www.abcam.com/ps/pdf/protocols/stripping for reprobing.pdf>

110. Seta, L., Baldino, N., Gabriele, D., Lupi, F. R. & de Cindio, B. Rheology and adsorption behaviour of β-casein and β-lactoglobulin mixed layers at the sunflower oil/water interface. Colloids Surfaces, A Physicochem. Eng. Asp. 441, 669–677 (2014).

111. Fang, F. & Szleifer, I. Kinetics and thermodynamics of protein adsorption: a

generalized molecular theoretical approach. Biophys. J. 80, 2568–2589 (2001).

112. Bromley, E. H. C., Krebs, M. R. H. & Donald, A. M. Mechanisms of structure formation in particulate gels of ??-lactoglobulin formed near the isoelectric point. Eur. Phys. J. E 21, 145–152 (2006).

113. Tahmasebi, E., Bozorgi, M. & Khukhan, K. A kinetic Study in the changes of SDS-PAGE profile of whey proteins during storage at different temperatures. 2, 65–70 (2014).

114. Demanèche, S., Chapel, J. P., Monrozier, L. J. & Quiquampoix, H. Dissimilar pH-dependent adsorption features of bovine serum albumin and α-chymotrypsin on mica probed by AFM. Colloids Surfaces B Biointerfaces 70, 226–231 (2009).

115. Paulsson, M. & Dejmek, P. Thermal denaturation of whey proteins in mixtures with caseins studied by differential scanning calorimetry. J. Dairy Sci. 73, 590–600

(1990).

116. Mathes, J. M. Protein Adsorption to Vial Surfaces – Quantification , Structural and Mechanistic Studies. Adsorpt. J. Int. Adsorpt. Soc. 1–233 (2010).

117. Dutta, D. S., Chattoraj, D. K., Chattopadhyay, P. & Das, K. P. Excess adsorption of biomolecules on soft surfaces : Adsorption of DNA , proteins and lactose on fatty surfaces. 2013, 40–47 (2013).

118. Pinholt, C., Hartvig, R. A., Medlicott, N. J. & Jorgensen, L. The importance of interfaces in protein drug delivery - why is protein adsorption of interest in

pharmaceutical formulations? Expert Opin. Drug Deliv. 8, 949–964 (2011).

119. Nail, S. L., Jiang, S., Chongprasert, S. & Knopp, S. A. Development and manufacture of Protein Pharmaceuticals. Fundamentals of Freeze-Drying 14, (2002).

120. Maes, K., Smolders, I., Michotte, Y. & Van Eeckhaut, A. Strategies to reduce aspecific adsorption of peptides and proteins in liquid chromatography-mass spectrometry based bioanalyses: An overview. Journal of Chromatography A 1358, 1–13 (2014).

121. Murray, B. & Deshaires, C. Monitoring Protein Fouling of Metal Surfaces via a

71

Quartz Crystal Microbalance. J. Colloid Interface Sci. 227, 32–41 (2000).

122. Aceti, D. J. et al. Role of nucleic acid and protein manipulation technologies in high-throughput structural biology efforts. in 8, 469–496 (Wiley-VCH Verlag

GmbH, 2003).

72

Appendix A

73

1. Time Dependent Silver Staining

Silver staining though a very sensitive protein stain, its use is often limited by lack

of linearity and difficult to control background staining. One way to avoid these problems

is to monitor the staining process and identifying an optimum time to quench staining

reaction for each constituent protein in a sample in my case of milk proteins, I observed

that β casein and β lactoglobulin were the bands that appeared quickly but also lost

linearity before other bands appeared. Hence, I tried to quantify them using earlier

frames of the staining video.

Figure S1: Time dependent staining for Figure 2-4 and 2-5

Experiment - 1

Time

(min)

BSA α Casein β Casein β LG α LA

0

3

4

5

6

7

8

74

9

10

Experiment - 2

Time

(min)

BSA α Casein β Casein β LG α LA

0

7

8

9

10

11

12

13

14

Experiment - 3

75

Time

(min)

BSA α Casein β Casein β LG α LA

5

6

7.00

7.22

8

9

10

11

12

76

2. Standard curves from time dependent silver staining

Since it was evident that β casein band saturates and loses linearity quickly as

compared to other milk proteins, a video was recorded for each gel staining so that

staining could continue even after the β casein band had appeared. The video of silver

staining was recorded and a frame was selected at an average interval on 1 min. The

frame was tested for the slope of the standard curve with regards to β casein, and the

frame with the greatest slope was chosen for the standard curve.

β Casein Standard Curve for Figure 4.1

For this experiment, the time chosen was 9 min.

77

β Casein Standard Curve for Figure 4.2

For this experiment, the time chosen was 7 min.

β Casein Standard Curve for Figure 4.4

Since figure 4.4 had two gels that have samples on it, there were two gels that have their

own β casein standards for quantification

Gel 1:

For this gel, the time chosen was 7 min.

78

Gel 2:

For this gel, the time chosen was 6 min.