1

Tools for Improved

Protein Mass Spec Sample Preparation

Mourad FERHAT, Ph.D.

15/03/2017

Promega Francemourad.ferhat@promega.com

Promega Corporation

Manufacturer of reagents, kits and integrated systems for life science market

Promega Headquarters Madison, WI

Founded in 1978

1,300 employees in 15 countries

Over 3,500 products

ISO 13485 certified

~ 750 patents

Operations in:

San Luis Obispo, CA

Sunnyvale, CA

Seoul and Shanghai

2

Protein fractionation

Mass spec analysis

Protein mass spec sample preparationCommon shortcomings

peptides

Incomplete digestion

Trypsin is not suitable for analysis

Poor peptide recoveryLong and laborious

sample prep procedure

Poor protein extraction fromtissues

Protein extraction

Inadequate instrument performancemonitoring

Protein digestion

3

Enhanced Proteolysis with

Trypsin/Lys-C Mix

4

Minimized missed cleavages

Increased tolerance to trypsin inhibiting agents

Trypsin, Sequencing Grade

The highest digestion efficiency Tolerance to protease inhibiting agents

Digestion efficiency

Higher digestion efficiency Higher purity (TPCK treatment + affinity

purification) Resistant to autolysis (Lysine residues are

modified by reductive methylation)

Overall good performance

Enhancing of trypsin performance

5

Trypsin Gold, Mass Spec Grade

Trypsin/Lys-C mix

What is the nature of incomplete proteolysis in trypsin digests?

Overnight trypsin digest of yeast protein extract

22.2%missedcleavages

Missed R 3.6%

Missed K 18.6%

2.6

4

Majority of missedcleavages occurs atlysine sites.

Missed cleavages

Trypsin cleavage sites

NNNNR NNNNKNNNN

K

RK

K

K K

KK

6

R : Arginine K : Lysine

Solution: supplementing trypsin with Lys-C

Lys-C

NNNNK NNNN

Lysines are cleaved with high efficiency.

Lys-C is an ideal means to compensate for trypsin lysine cleavage inefficiency.

Trypsin

NNNN(R/K) NNNN

Lysines are cleaved less efficiently.

7

Enhanced proteolysis with Trypsin/Lys-C

Missed R 3.6%

Missed K 18.6%

Trypsin/Lys-C digest

3.6%4%

Overnight digestion at 37oC

Trypsin digest

8

Enhanced proteolysis with Trypsin/Lys-C

Missed R 3.6%

Missed K 18.6%

Trypsin digest Trypsin/Lys-C digest

9

3.6%4%

Trypsin/Lys-C eliminates majority of missed cleavage sites.

Overnight digestion at 37oC

Study #1: Analysis of FFPE skin tissue

21.5%

8.5%

Trypsin/Lys-C

Missed Cleavages, %

TrypsinTrypsin

Identified Peptides

Trypsin/Lys-C

Identified Proteins

TrypsinTrypsin/Lys-C

24% increase 10% increase2.5 fold drop

705887

165

10

182

Sample prep is difficult due to extensive protein crosslinking in FFPE tissue.

Courtesy of Chris Adams, Stanford U

Trypsin/Lys-C increased number of identified peptides and proteins in FFPE tissue.

Study #2: Developing biomarker quantitative assay for human plasma

Courtesy of Matt Szapacs, GSK

674099

Trypsin/Lys-C digest

9139

Trypsin/Lys-Cdigest

Trypsin digest

3743

Trypsin/Lys-C digest

1180

Trypsin/Lys-Cdigest

TrypsindigestTrypsin

digest Peptide peak area

Trypsin digest

SAP protein

145305

8976

2207 555

11

Trypsin/Lys-C provided conditions for more accurate quantitation of the targeted protein in plasma.

Study #3: Increased tolerance to trypsin inhibiting agents

Inhibitor: GuClInhibitor: proteaseinhibitor cocktail

1252

1495

Trypsin/Lys-C mix assures efficient proteolysis even if a proteinsample is contaminated with trypsin inhibiting agents.

12

Inhibitor Protease Missed cleavages

Protease inhibitorcocktail, 1X

Trypsin 44.4%

Trypsin/Lys-C 21.5%

GuCl, 0.5 MTrypsin 55.9%

Trypsin/Lys-C 24.6%

Digestion of yeast protein extract containing trypsin inhibiting agents

Missed (undigested) cleavage sites Identified proteins

Trypsin

Trypsin/Lys-C

Trypsin

13-20% increase

1364

1204

Trypsin/Lys-C

Advantages of Alternative Proteases

13

Alternative cleavage specificity

Activity under trypsin-inhibiting conditions

Alternative cleavage specificity and reactionconditions

14

Protease Cleavage site Property and application

Lys-C NNNNK NNN Active under denaturing conditions Digest proteolytically resistant proteins

Glu-C NNNNE NNN Used as trypsin alternative if trypsin cleavagesites have disadvantageous distribution

Asp-N NNNN DNNN

Arg-C NNNNR NNN(also cleave at K at a lesser degree)

Analysis of histone posttranslationalmodifications

Chymotrypsin NNNN(F/Y/W) NNN Digests hydrophobic proteins (i.e. membrane proteins)

Pepsin Nonspecific protease Works at low pH Used in HDX studies

Thermolysin Nonspecific protease Works at elevated temperature Digest proteolytically difficult proteins;

structural studies

Elastase Nonspecific protease Used to increase protein coverage

Pepsin and thermolysin are a better alternative for membrane proteins than trypsin

Fully digest membrane proteins

Low pH and high temperature used by these proteases help unfolding these proteins.

Case study: digestion of membrane proteins

Too few tryptic cleavage sites

Tight folding prevents trypsinaccess to cleavage sites

15

20

0

Bacteriorhodopsin coverage was dramatically increased whendigested with thermolysin and pepsin.

16

40

60

100

80

Pepsin

Thermolysin

Trypsin

Seq

ue

nce

cove

rage

,%

Bacteriorhodopsin

Coverage with trypsin

Coverage with pepsin

Bacteriorhodopsin sequence coverage

Increased protein sequence coverage with pepsinand thermolysin

IdeS – Immunoglobulin Degrading Enzyme fromStreptococcus pyogenes

IdeS is an IgG-specific protease. It cleaves IgG at a unique site below the hinge.

’

Fc/2 (+Glycans)

LC

Mass spec

30 min digestion

Fd’

IdeS

17

IdeS advantage for IgG characterization

Ready separation of IgG Fragments Rapid analysis of major protein modifications

IdeZ and IdeS protease cleavage specificity

We have recently added IdeZ protease to ourmass spec reagent portfolio. IdeZ offers furtherimprovement for IgG analysis.In contrast to IdeS, which preferentially cleaveshuman antibodies, IdeZ also efficiently cleavesmouse antibodies.

Characterization of Protein Glycosylation

19

PNGaseF

EndoH

Analysis of Glycoproteins with LC/MS and PNGase F

Asn-linked type glycans can be cleaved enzymatically by PNGase F :

The cleavage separate intact oligosaccharides from slightly modified proteins(Asn residues at the site of N-glycosylation are converted to Asp)

The deglycosylated peptides can be analyzed by mass spectrometry

20

Using EndoH and PNGase F to monitor protein

trafficking

Protein Digestion in Gel with MS Compatible

Surfactant

Promega Corporation 19

Increased peptide recovery

In-gel digestion and peptide extraction in a single 1h step

In-gel protein digestionAdvantages and challenges

Advantages of SDS-PAGE protein fractionation

Rapid removal of mass spec interfering impurities

Efficient reduction of sample complexity

23

Shortcomings of in-gel protein digestion

Inefficient peptide recovery from gel

Extensive peptide loss due to adsorption to a plastic ware

Lengthy and laborious procedure

In-gel protein digestionAdvantages and challenges

24

ProteaseMAX™ Surfactant

Cleavable bonds

Degradation by temperature or acid

LC/MS compatible compounds

Zwitterionic headHydrophobic tail+

Degradation Products

ProteaseMAX™ mass spec compatible surfactant

Mass spec compatible anionic surfactant

Cleavable bonds

Self-degradable mass spec compatible surfactant

ProteaseMAX™ is designed to self-degrade over the course of mass specprotein sample preparation onto mass spec innocuous compounds.

25

Peptide Increase in peptide recovery withProteaseMAX™, fold

AGGALCANGAVR 1.45QGDDGAALEVIEVHR 2.06

EHLPLPSEAGPTPCAPASFER 1.80

Improved peptide recovery

ProteaseMAX™ increases peptide recovery from gel.

MALDI-TOF spectrum of HTR1A protein digested in gel

Peptides recovered with ProteaseMAX™

Peptides recovered in conventional digestion

Saveliev et al. Analytical Chemistry 2013, 85 (2), pp 907–914.

26

Minimized peptide adsorption to plastic ware

27

Peptide Increase in soluble peptide with ProteaseMAX (fold)

PLSRTLSVAAK 16.6

TTYADFIASGRTGRRNAIHD 9.2

AAKIQASFRGHMARKK 4.6

EPPLSQEAFADLWKK 2.05

Saveliev et al. Analytical Chemistry 2013, 85 (2), pp 907–914.

ProteaseMAX™ minimizes peptide adsorption to a plastic ware.

Enhanced analysis of a complex protein mixture withProteaseMAX™-assisted in-gel digestion

Gel-LC Analysis of Mouse Protein Extract

Courtesy of Dr. Chris Adams, Stanford U

ProteaseMAX™ increases number of peptide and proteinidentifications in a cell extract digested in gel.

28

Conventional In-gel Protein Digestion

Peptide extraction(1.5 – 2 h)

Mass spec analysis Mass spec analysis

29

In-gel Protein Digestion withProteaseMAX™

Digestion/extraction step(1 h)

Protein digestion and peptide extraction are complete in a single 1 h step.

12-18 h

Streamlined and rapid in-gel digestion withProteaseMAX™

Protein Extraction from Tissues with MS

Compatible Surfactant

Promega Corporation 27

MS compatible SDS analog for tissue proteomics

Efficient extraction of hydrophobic proteins

Surfactant 3273 – MS compatible SDS analog fortissue proteomics

Surfactant 3273

Degradation by a strong acid

Cleavable bonds

Mass spec compatible anionic surfactant

Degradation Products

Hydrophobic tail Zwitterionic head

+

LC/MS compatible compounds

31

3273 is designed for efficient protein extraction from tissues and other biological samples and solubilization of protein pellets.

Enhanced protein extracting and solubilizing capability Tolerates harsh treatment, including boiling

Improved protein extraction from pig heart withsurfactant 3273

Protein IDs in pig liver extractsTotal extracted protein

SDS-PAGE

Chang et al. J. Proteome Res. 2015, 14 (3), pp 1587–1599.

RapiGest

Control

3273

Control

32

Total membrane protein IDs in tissue extracts

3273

3273 enhances protein extraction from animal tissues. Protein extraction efficiency is comparable to SDS. Number of recovered membrane proteins is significantly increased.

Reference MS Protein Materials

30

Validated test material for sample prep optimization

Standards for monitoring all key LC and MS performance parameters

Highly complex reference protein material for:

Mass spec instrument performance monitoring

Sample preparation method development

Features

Compatible with LC/MS

Pre-processed for immediate use

Lot to lot consistency in protein composition and abundance

Provided in intact and pre-digested formats.

MS-compatible whole cell protein extractsModel proteomic material

K562 human cells

Yeast

Reference Protein Materials address the critical question:Do my mass spec instrument, reagents and method work properly?

34

Mass spec instrument performance monitoring

RT: 0.00 - 106.14 SM: 5G

0 30 40 50 60 70 80 90 100

Time (min)

40

20

0

80

60

100

0

40

20

80

60

100

40

20

0

80

60

10087.6345.71

38.7423.45 82.0432.20 35.72 79.9543.26 52.11 58.3726.04 61.02 77.63 90.0665.57 98.66

21.09

15.17 19.385.10

44.31

87.3730.83

37.51

41.9722.59 81.30 81.9649.48 50.78 57.2021.37 89.7775.56 76.69

97.6218.5711.088.74

38.22

45.1087.9122.92

31.60 82.6635.2025.48 58.4548.88 82.4565.43

72.4396.42 98.61

20.61

14.88 18.94

10 20

8.10

st

35

1 week

nd2 week

3rd week

Detecting deterioration of an instrument performance in a timelyManner (1 µg of human predigested protein extract)

Re

lati

ve a

bu

nd

ance

Example of compromised instrument performance

10 20 30 40 50 60 70 80

40

30

20

10

0

42.1858.19 90.22

86.1546.10 56.29

59.8135.74

83.5729.6163.33 63.41 70.5054.44 75.9225.88

92.56 97.95 1

90 100

23.9111.10 20.39

Courtesy by MS BioWorks, Ann Arbor, MI

Good quality chromatogram (an instrument properly works)

Time(min)

Poor quality chromatogram (an instrument needs maintenance)

Peptide ionization and retention times are compromised

Detecting deterioration of an instrument performance in a timelymanner.

Re

lati

veab

un

dan

ceR

ela

tive

abu

nd

ance

40

30

20

10

0

40.09 51.15

60.3328.07 32.39 92.54 92.7866.58 74.10 82.28

12.00 25.9823.17

36

6x5 peptide mixProduct concept

Isomer # Sequence MW M

1 LLSLGAGEFK 1072.67318 0.00

2 LLSLGAGEFK 1062.64598 10.03

3 LLSLGAGEFK 1055.62878 7.02

4 LLSLGAGEFK 1048.61158 7.02

5 LLSLGAGEFK 1041.59448 7.02

most hydrophilic peptide

MS

Peak

Inte

nsi

ty

LC Chromatogram C18 LC Gradient (increasing hydrophobicity)

most hydrophobic peptideIn

ten

sity

Peptide Retention Time

Linear Dynamic range

m/z

Six peptides. Each peptide is represented by five isotopologues mixed within linear concentration

dynamic range.

37

A mixture of 6x5 = 30 peptides for completemonitoring of LC-MS/MS parameters

Each peptide has five chromatographically indistinguishable isotopologues, with abundances spanning four orders of magnitude. Bolded amino acids (in red) are uniformly labeled with stable 13C and 15N atoms.

38

Beri et al. Analytical Chemistry 2015, 87, 11635−11640http://pubs.acs.org/doi/abs/10.1021/acs.analchem.5b04121

Mass spec products to be launched soonRapid Trypsin

2. Heat increases enzymatic activity.

1. Heat induces protein unfolding providing easy protease access to cleavage sites.

Heat12-18 hdigestion at 37oC

Heat advantages for proteolysis

Digested peptides Digested peptides

IgG digest IgG digest

Rapid Trypsin is a thermostable formulation of trypsin. It rapidly digests proteins at high temperature.

Conventional Protein Digestion

Denaturation

Reduction

Alkylation

39

30 min digestion with no need for reduction and alkylation.

Rapid digestion with Rapid Trypsin

30 min at 70oC

Mass spec products to be launched soonRapid Trypsin

Rapid Trypsin is not for anytrypsin customer because ithas certain shortcomings.However, it perfectly fits the needs of the customers working in:

Drug discovery and pharmacokinetics fields

Proteomics

Clinical research12-18 hdigestion at 37oC

Digested peptides Digested peptides

IgG digest IgG digest

30 min digestion with no need for reduction and alkylation.

Rapid digestion with Rapid Trypsin

30 min at 70oC

Rapid Trypsin is a thermostable formulation of trypsin. It rapidly digests proteins at high temperature.

Conventional Protein Digestion

Denaturation

Reduction

Alkylation

40

Mass spec products to be launched soonLow pH digestion kit for biotherapeutic protein characterization

This kit is designed for the most accurate characterization of biotherapeutic proteins with LC/MS and UV-HPLC.

41

Mass spec products to be launched soon

Digestion

Digested peptides

42

Denaturation

Reduction

Alkylation

Low pH digestion kit for biotherapeutic protein characterization

Digestion and other sample prep steps induce artificial posttranslational modifications including:

Deamidation

Disulfide bond scrambling

Oxidation

These modifications compromise characterization of biotherapeutic proteins.

Mass spec products to be launched soonLow pH digestion kit for biotherapeutic protein characterization

Digestion and other sample prep steps induce artificial posttranslational modifications including:

Deamidation

Disulfide bond scrambling

Oxidation

Digestion

Digested peptides

Denaturation

Reduction

Alkylation

Conventional digest

Low pH digest

peptide

Deamidation is eliminated

43

Re

lati

ve

abu

nd

ance

These modifications compromise characterization of biotherapeutic proteins.

We have suppressed artificial PTMs by decreasing reaction pH from conventional alkaline range to acidic range (it was a very tricky thing to do!). This has significantly improved quality of biotherapeutic protein analysis.

Suppression of deamidation in RituximabGLEWIGAIYPGnGDTSYNQK peptide

Deamidated

Mass spec products to be launched soonRapid Trypsin and Low pH digestion kit

Both products have been extensively tested by customers. Customers are happy with the product performance and waiting for the product launch.

44