2D Gel Electrophoresis

By: Ashish C PatelAssistant Professor

Vet College, AAU, Anand

• 2-D electrophoresis is a powerful and widely used method for the analysis of complex protein mixtures extracted from cells, tissues, or other biological samples.

• It is the method available which is capable of simultaneously separating thousands of proteins.

• This technique separate proteins in two steps, according to two independent properties:

First-dimension is isoelectric focusing (IEF), which separates proteins according to their isoelectric points (pI);

Second-dimension is SDS-polyacrylamide gel electrophoresis (SDS-PAGE), which separates proteins according to their molecular weights (MW).

• In this way, complex mixtures consisted of thousands of different proteins can be resolved and the relative amount of each protein can be determined.

• Each spot on the resulting two-dimensional gel potentially corresponds to a single protein species in the sample.

• Thousands of different proteins can be separated and information such as the protein pI, the apparent molecular weight, and the amount of each protein can be obtained.

• At the very beginning of the 70s, two high-performance electrophoretic separations of proteins were available: i) zone electrophoresis of proteins in the presence of SDS, as described in its almost final form by Laemmli, a technique that instantly became very popular, and still is, and ii) denaturing isoelecric focusing, as described by Gronow and Griffith.

• As these two techniques used completely independent separation parameters, it is not surprising that it was soon tried to couple them.

• Two-dimensional electrophoresis was first introduced by O’Farrell in 1975.

Principle:• In 2D GE proteins are separated as per isoelectric point and protein mass.• Separation of the proteins by isoelectric point is called isoelectric focusing (IEF).

When a gradient of pH is applied to a gel and an electric potential is applied across the gel, making one end more positive than the other.

• At all pH values other than their isoelectric point, proteins will be charged. If they are positively charged, they will be pulled towards the negative end of the gel and if they are negatively charged they will be pulled to the positive end of the gel. The proteins applied in the first dimension will move along the gel and will accumulate at their isoelectric point; that is, the point at which the overall charge on the protein is 0 (a neutral charge).

• In separating the proteins by mass, the gel treated with sodium dodecyl sulfate (SDS) along with other reagents (SDS-PAGE in 1-D). This denatures the proteins (that is, it unfolds them into long, straight molecules) and binds a number of SDS molecules roughly proportional to the protein's length. Because a protein's length (when unfolded) is roughly proportional to its mass, Since the SDS molecules are negatively charged, the result of this is that all of the proteins will have approximately the same mass-to-charge ratio as each other.

• In addition, proteins will not migrate when they have no charge (a result of the isoelectric focusing step) therefore the coating of the protein in SDS (negatively charged) allows migration of the proteins in the second dimension.

• In the second dimension, an electric potential is again applied, but at a 90 degree angle from the first field. The proteins will be attracted to the more positive side of the gel (because SDS is negatively charged) proportionally to their mass-to-charge ratio.

• The gel therefore acts like a molecular sieve when the current is applied, separating the proteins on the basis of their molecular weight with larger proteins being retained higher in the gel and smaller proteins being able to pass through the sieve and reach lower regions of the gel.

Overview

1. Sample preparation

2. Isoelectric focusing (first dimension)

3. SDS-PAGE (second dimension)

4. Visualization of proteins spots

5. Identification of protein spots

Steps in 2D-Gel Electrophoresis

1. Sample preparation• Must break all non-covalent protein-protein, protein-DNA, protein-

lipid interactions, disrupt S-S bonds• Must prevent proteolysis, accidental phosphorylation, oxidation,

cleavage, deamidation• Must remove substances that might interfere with separation

process such as salts, polar detergents (SDS), lipids, polysaccharides, nucleic acids

• Must try to keep proteins soluble during both phases of electrophoresis process

Sample preparation……….

• Protein Solubilization– 8 M Urea (neutral chaotrope)– 4% CHAPS (zwitterionic detergent)– 2-20 mM Tris base (for buffering)– 5-20 mM DTT (to reduce disulfides)– Carrier ampholytes or IPG buffer (up to 2% v/v) to enhance

protein solubility and reduce charge-charge interactions• Protease inhibitors

– PMSF(PhenylmethaneSulfonyl Flouride), Pefabloc, EDTA,, leupeptin, Aproteinin, Pepstatin

• Contaminant removal– Filtration, Centrifugation, Chromatography, Solvent Extraction

1st dimension: Isoelectric Focusing

• In a pH gradient and under the influence of an electric field, a protein will move to the position in the gradient where its net charge is zero.

• A protein with a net positive charge will migrate toward the cathode, becoming progressively less positively charged as it moves through the pH gradient until it reaches its pI.

• A protein with a net negative charge will migrate toward the anode, becoming less negatively charged until it also reaches zero net charge.

• If a protein should diffuse away from its pI, it immediately gains charge and migrates back. This is the focusing effect which allows proteins to be separated on the basis of very small charge differences.

• The resolution is determined by the slope of the pH gradient and the electric field strength so, IEF is therefore performed at high voltages (typically in excess of 1000 V).

• When the proteins have reached their final positions in the pH gradient, there is very little ionic movement in the system, resulting in a very low final current.

• The original method for first-dimension IEF depended on ampholyte-generated pH gradients in cylindrical polyacrylamide gels cast in glass rods or tubes. Now it is replaced by DryStrip gels.

Advantages of DryStrip gels include:• The first-dimension separation is more reproducible because the

covalently fixed gradient cannot drift.• Plastic-backed DryStrip gels are easy to handle. They can be picked up at

either end with forceps or gloved fingers.• The plastic support film prevents the gels from stretching or breaking.• More acidic and basic proteins can be separated.• The sample can be introduced into the DryStrip gel during rehydration.• DryStrip gels eliminate the need to handle toxic acrylamide monomers. • Immobilized pH gradients and precise lengths ensure high reproducibility

and reliable gel-to-gel comparisons.

1st dimension: Isoelectric Focusing• Isoelectric Point (pI):

– pH at which a protein has a neutral charge; loss or gain of protons H+ in a pH gradient (In a pH below their pI, proteins carry a net positive charge and in a pH above their pI, they carry a net negative charge)

• Requires very high voltages (10000V)• Requires a long period of time (10h)• Degree of resolution determined by slope of pH gradient and electric

field strength• Uses ampholytes to establish pH gradient• IPG strips:

– An immobilized pH gradient (IPG) is made by covalently integrating acrylamide and variable pH ampholytes at time of gel casting, Stable gradients

Components of rehydration solution

• The choice of the rehydration solution for the sample will depend on its specific protein solubility requirements.

• A typical solution generally contains urea, nonionic or zwitterionic detergent, DeStreak Reagent or DTT, the appropriate Pharmalyte or IPG Buffer and a tracking dye.

• Urea solubilizes and denatures proteins, unfolding them to expose internal ionizable amino acids. Commonly, 8 M urea is used, but the concentration can be increased to 9 or 9.8 M

• Thiourea, in addition to urea, can be used to further improve protein solubilization, particularly for hydrophobic proteins . When using both, the recommended concentration of urea is 7 M and that of thiourea 2 M.

• Detergent solubilizes hydrophobic proteins and minimizes protein aggregation. The detergent must have zero net charge—use only nonionic or zwitterionic detergents. CHAPS, Triton X-100, or NP-40 in the range of 0.5 to 4% are most commonly used.

Isoelectric Focusing…………….

Rehydrate IPGstrip & apply

protein sample

Place IPG stripin IEF apparatus

and apply current

Equilibration, reduction and alkylationSDSUreaDTTIodoacetamide

• SDS-PAGE is an electrophoretic method for separating polypeptides according to their molecular weights. The technique is performed in polyacrylamide gels containing sodium dodecyl sulfate (SDS).

• SDS is an anionic detergent. SDS masks the charge of the proteins themselves net negative charge per unit mass.

• Besides SDS, a reducing agent such as DTT is also added to break any disulfide bonds present in the proteins.

• When proteins are treated with both SDS and a reducing agent, the degree of electrophoretic separation within a polyacrylamide gel depends largely on the molecular weight of the protein.

• In fact, there is an approximately linear relationship between the logarithm of the molecular weight and the relative distance of migration of the SDS-polypeptide complex.

2nd Dimension (SDS-PAGE)

• Separation of proteins on basis of MW, not pI• Requires modest voltages (200V)• Requires a shorter period of time (2h)• Presence of SDS is critical to disrupting structure and

making mobility ~ 1/MW• Degree of resolution determined by %acrylamide &

electric field strength

Steps of SDS PAGE

1) Preparing the system for second-dimension electrophoresis2) Equilibrating the gel(s) in SDS equilibration buffer3) Placing the equilibrated gel on the SDS gel4) ElectrophoresisEquilibrating the gels: • It is important to proceed immediately to gel equilibration, unless

the IPG strip is being frozen (at -60 °C or below) for future analysis. • Equilibration is always performed immediately prior to the second-

dimension run, never before storage of the DryStrip gels.• The second-dimension gel itself should be prepared and ready to

accept the DryStrip gel before beginning the equilibration protocol.

Equilibration solution components• The equilibration step saturates the gel with the SDS buffer system required

for the second dimension separation. • The equilibration solution contains buffer, urea, glycerol, reductant, SDS, and

dye. An additional equilibration step replaces the reductant with iodoacetamide.

• Equilibration buffer (75 mM Tris-HCl, pH 8.8) maintains the DryStrip gel in a pH range appropriate for electrophoresis.

• Urea (6 M) together with glycerol reduces the effects of electroendosmosis by increasing the viscosity of the buffer.

• Glycerol (30%) together with urea reduces electroendosmosis and improves transfer of proteins from the first to the second dimension.

• Dithiothreitol (DTT) preserves the fully reduced state of denatured, unalkylated proteins.

• Sodium dodecyl sulfate (SDS) denatures proteins and forms negatively charged protein-SDS complexes.

• iodoacetamide alkylates thiol groups on proteins, preventing their reoxidation during electrophoresis.

• iodoacetamide also alkylates residual DTT to prevent point streaking and other silver-staining artifacts .

• Tracking dye (bromophenol blue) allows monitoring of the progress of electrophoresis.

• The most commonly used buffer system for second-dimension SDS-PAGE is the Tris-glycine system described by Laemmli.

• This buffer system separates proteins at high pH, which confers the advantage of minimal protein aggregation and clean separation even at relatively heavy protein loads.

Procedure:Equilibration1. Place 2 mL of Equilibration buffer I on each thawed strip in gel tray. (This

step reduces the proteins.)2. Place the lid on the gel tray. Place tray on shaker and shake gently for 10minutes.3. Remove equilibration buffer I from the strips and drain into non-

chlorinated waste under the hood. Hold the flat side (not slanted side) down to discard buffer and do not touch the strips.

4. Repeat process with equilibration buffer II. (This step alkylates proteins.)

SDS Running Buffer1. Rinse out the graduated cylinder specified for SDS running buffer with

MilliQ H2O.2. Fill to 100 mL with SDS running buffer (10x) and dilute to 1000 mL with

MilliQ H2O.3. Remove bubbles from top of beaker.

Preparation of Gels1. While tray is shaking, get out the gels. The liquid within the packaging

has sodium azide, so handle with caution. Remove the white strip and green comb from the gels.

2. Rinse gels with MilliQ H2O from bottle located in gel room. Be sure to thoroughly rinse the well and the molecular weight marker well.

3. Blot with filter paper squares to remove excess water (Do not touch surface of gel inside well). Be careful not to disturb molecular weight marker well. You do not want water in area where strip will be placed.

Loading of Gels1. Loosen the lid and heat the overlay agarose in microwave until

melted. Heat in 10 second (or less) increments. ~30 seconds total.2. Pick the strip up with tweezers on the “+” side of the strip. Slide the

gel strip onto the edge of the gel tray.3. Dip the strip in SDS running buffer 5 times and then allow excess

buffer to drain off onto a paper towel.

4. Lay down gel and place strip gel side up onto the top of the gel casette with the ”+” end toward the molecular weight marker well. Leave the strip at the top aligned above well.

5. With the gel box upright (lean it against a large tip box), add 1 mL of overlay agarose to well and MW well. Make sure that no bubbles form. If they do, remove them with the end of a pipette tip.

6. Push the strip down into the overlay agarose. Push one side down 1st and slowly push other side down at an angle to prevent formation of bubbles. Make sure the strip lays flat against the gel.

7. Add 2 μL of appropriate molecular weight marker to the molecular weight well. Add marker as deep as possible in the well to prevent the molecular weight marker from spilling over into the other area of the gel.

8. Place gel in gel box. Be careful to hold the gel level while placing it in the gel box.

9. Once agarose is solidified, fill gel box with the SDS running buffer to the fill line (don’t pour directly on agarose). You want to fill the cassette chambers with running buffer as well.

Gel Box Run1. Place the lid on the gel box (red to red; black to black).2. Plug the gel box into the voltage box (red to red; black to blue).3. Turn on the voltage to 60 Volts for 15 minutes.4. Turn the voltage up to 200 Volts for the remainder of the run (45-60

minutes).After the Gel Run1. Unplug gel box from voltage box.2. Remove cassette from gel box, drain SDS running buffer.3. Crack all 4 joints of the cassette with a green comb.4. Fill plastic bottom of gel container with MilliQ H2O.5. Place gel in tray by turning cassette upside down and placing gently

into water.6. Follow the instructions on the wall of the gel room for the

appropriate stain.

IPG strip-pressed down into the SDS-PAGE gel

Positive electrode

Negative electrode

Similar mw but different pI

Similar pI but different mw

pH 3 4 5 6 7 8 9 10

4. Detection/Visualization• Coomassie Stain (100 ng to 10 mg protein)• Silver Stain (1 ng to 1 mg protein)• Fluorescent (Sypro Ruby) Stain (1 ng & up)

In gel digestion

5. Protein Identification

Phosphoimager for32P and 35S labelled

1D or 2D gels

Fluoroimager forSYPRO labelled1D or 2D gels

Densitometer orPhoto Scanner

Imaging

Melanie (http://ca.expasy.org/melanieImageMaster 2D (Amersham)PDQuest (BioRad )

Analysis

Excision of spots

MSMS/MS

Protein detection and image analysis

• This step plays a crucial role, as i) only what is detected can be further analyzed and ii) quantitative variations observed at this stage are the basis to select the few spots of interest, in comparative studies, that will be the only ones processed for further analysis with mass spectrometry.

• Detection with organic dyes can be summarized in one single process, colloidal Coomassie Blue staining, which has really become a reference standard.

• Although the sensitivity is moderate and homogeneity are good and compatibility with mass spectrometry is excellent.

• Silver staining is much more sensitive but less homogeneous, because of its delicate mechanism, and its compatibility with mass spectrometry is problematic.

• The consequence of the presence of formaldehyde at the image development step, formaldehyde-free silver staining protocols have been recently proposed.

• Protein detection by fluorescence give good sensitivity and also good compatibility with mass spectrometry .

• Other modes of detection are environment-sensitive probes, noncovalent binding and covalent binding.

• The use of chemically related, reactive fluorescent probes differing mainly by their excitation and emission wavelengths allows to perform multiplexing of samples on 2D gels.

• This multiplexing process solves in turn two difficult problems in the comparative analysis of gel images, namely the assignment of small positional differences and taking into account moderate quantitative changes.

• Steps in proteome informatics for 2-DE are:Image acquisition: This prepares each raw acquisition for subsequent

comparative analysis. After scanning, the images are pre-processed by cropping (manual delineation), noise suppression, and background subtraction (e.g., with mathematical morphology or smooth polynomial surface fitting).

An image capture device is required, for which there are three main categories:

• Flatbed scanner: This mechanically sweeps a standard charge-coupled device (CCD) under the gel and can be used to obtain 12–16 bits of greyscale or colour densitometry from visible light stains. Noise can be an issue due to size and cooling restrictions. Calibration is often required to provide linearity. flatbed scanners are typically the least expensive offerings. Examples: ImageScanner (GE Healthcare), ProteomeScan (Syngene) and GS-800 (Biorad).

• CCD camera: Since the sensor is fixed, its greater size and cooling provides a dramatic improvement in noise. Different filters and transillumination options allow a wide range of stains to be imaged, including visible light, fluorescent, reverse, chemiluminescent, and radioactive signals. However, the fixed sensor limits image resolution. Examples: LAS (Fuji Photo Film), ImageQuant (GE Healthcare), Dyversity (Syngene), BioSpectrum2D (UVP) and VersaDoc (Biorad).

• Laser scanner: Photomultiplier detectors are combined with laser light and optical or mechanical scanning to pass an excitation beam over each target pixel. While slower than CCD cameras, spatial resolution is excellent. FLA (Fuji Photo Film), Typhoon (GE Healthcare) and Pharos FX (Biorad).

Conventional analysis (Spot Detection Spot Matching): ≫ Each protein spot is delineated and its volume quantified. The spots are segmented first by the watershed transform, where spots are slowly immersed in water. Point pattern matching is then employed to match the spots between gels, which finds the closest spot correspondence between a point pattern (source spot list) and a target point set (reference spot list).

Image-based analysis (Gel alignment Consensus Spot Modelling): ≫ With current techniques, a “reference” gel is chosen and the other “source” gels are aligned to it in pair-wise fashion. A similarity measure which quantifies the quality of alignment between the warped source gel and the reference gel. The aim is to automatically find the optimal transformation that maximizes the similarity measure. Spot detection is then performed on an image, which is then propagated to each individual gel for spot quantification.

Differential analysis : At this stage, we have a list of spots, and for each spot, a quantified abundance in each gel. The abundances are first normalized to remove systemic biases between gels and between channels in DIGE gels. Variance stabilization can then be employed to remove the dependence between the mean abundance of a protein and its variance. Significance tests are then performed to obtain p-values for rejecting the null hypothesis that the mean spot abundance between groups is unregulated.

Advanced techniques : Since multiple hypothesis testing leads to a large number of false positives, it is essential to control the False Discovery Rate (FDR). The FDR is the estimated percentage of false positives within the detected differential expression rather than within the set of tests as a whole. Power analysis is also estimates the false negative rate that determines the optimal sample size needed to detect a specific fold change to a particular confidence level.

Applications

• The proteomics analysis reported here shows that a major cellular response to oxidative stress is the modification of several peroxiredoxins. An acidic form of the peroxiredoxins appeared to be systematically increased under oxidative stress conditions due to post transcriptional modifications.

• Peroxiredoxin 2 and 3 spots in Jurkat cells. cells were separated by two-dimensional electrophoresis. The peroxiredoxin spots (indicated by arrows) were identified by mass spectrometry.

• The cells were either control cells (A) or cells treated with 75 M BHP for 1 h (B). increase in the acidic peroxiredoxin spots under oxidative stress, and the corresponding decrease in the basic spot under BHP treatment.

1.

• Peroxiredoxin spots under various cell injury conditions.

• The peroxiredoxin spots (indicated by arrows) were identified by mass spectrometry. The cells were cultured under normal conditions (A), submitted to oxidative stress with 75 M BHP for 30 min (B) or 14 milliunits/ml glucose oxidase for 18 h (C), or treated with 1 M daunomycin for 18 h (D). The increase in the acidic spots is correlated with oxidative stress.

Protein Identification by Mass Profile Fingerprinting

• Due to the high resolution of 2D gels, very simple and cheap MS process can be used to identify a protein from a 2D gel.

• For example, the old peptide mass fingerprinting method, which is fairly cheap, fast, and can be carried out on low-price TOF MS, works only with 2D gel-separated proteins, and will never work with any other technique of less resolving power.

• We can identify proteins at the sub-microgram level without sequence determination by chemical degradation.

• The protein, usually isolated by one- or two-dimensional gel electrophoresis, is digested by enzymatic or chemical means and the masses of the resulting peptides are determined by mass spectrometry.

• The resulting mass profile, i.e., the list of the molecular masses of peptides produced by the digestion, serves as a fingerprint which uniquely defines a particular protein.

• This fingerprint may be used to search the database of known sequences to find proteins with a similar profile. This provides a rapid and sensitive link between genomic sequences and 2D gel electrophoresis mapping of cellular proteins.

2D gel-based proteomics is widely used in areas where large series of samples are the norm, for example in toxicology

• 2-DE is used to find an association between decreased calcium-binding protein (calbindin-D 28 kDa), urinary calcium wasting and intratubular corticomedullary calcifications in rat kidney .

• They show that in dogs and monkeys, which are generally devoid of cyclosporine A (CsA)-mediated nephrotoxicity b/c renal calbindin levels not affected by the CsA treatment

• whereas in CsA-treated human kidney-transplant recipients with renal vascular or tubular toxicity, a marked decrease in renal calbindin-D 28 kDa protein level was found in most of the kidney biopsy sections.

• It suggest that calbindin is a marker for CsA-nephrotoxicity. • The discovery of calbindin-D 28 kDa being involved in CsA toxicity has evolved

from the application of 2-DE and has not been reported previously, proving that proteomics can provide essential information in mechanistic toxicology.

2D gel-based proteomics in bacterial proteomics• 2D gel-based proteomics is also widely used in bacterial proteomics, when the

complexity of the sample is low enough.• (A) Theoretical proteome of B. subtilis showing the distribution of all 4100

predicted proteins according to their isolelectric points and molecular weights. • (B) B. subtilis master 2-D gel for cytoplasmic proteins which are separated in the

standard pH range 4–7 (right image) and in the alkaline pH range 7–12 (left image). In the master 2-D gel 519 proteins are labeled that were identified in the pH range 4–7. In addition, 174 proteins were identified in the narrow range pH gradients (pH 4–5, 4.5–5.5, 5–6, and 5.5– 6.7) and 52 proteins in the alkaline pH range 7–12.

• Cytoplasmic proteins were harvested from B. subtilis wild type cells grown in Belitsky minimal medium at an OD500 of 0.4 and separated by 2-D GE.

Use in immunoproteomics

• 2 D GE also used in Immunoproteomics, where it is the immune response of patients that is probed at a proteomic level.

• 2D GE maps of proteins from Chlamydia trachomatis were probed with sera from 17 seropositive patients with genital inflammatory disease.

• Immunoblot patterns (comprising 28 to 2 spots, average 14.8) were different for each patient; however, antibodies against a spot-cluster due to the chlamydia-specific antigen outer membrane protein-2 (OMP2) were observed in all sera.

• The next most frequent group of antibodies (15/17; 88%) recognized the hsp60 like protein, described as immunopathogenic in chlamydial infections.

• A novel outer membrane protein (OmpB) and, interestingly, five conserved bacterial proteins: RNA polymerase alpha-subunit, ribosomal protein S1, protein elongation factor EF-Tu, putative stress-induced protease of the HtrA family, and ribosomal protein L7/L12. These proteins were shown to confer protective immunity in other bacterial infections.

2D gels in post-translational modifications

• 2D gels are also very appropriate when post-translational modifications are studied.

• many post translational modifications do alter the pI and/or the MW of the proteins and thus induce position shifts in 2D gels. This is true for example for phosphorylation, glycosylation, but also more delicate modifications such as glutathionylation, or more forgotten modifications such as protein cleavage.

Symptom Possible cause Remedy

No distinct spots are visible

Sample is insufficient. Increase the amount of sample applied.

Sample contains impurities that prevent focusing.

Increase the focusing time or modify thesample preparation method

The pH gradient isincorrectly oriented.

The “+” end of the DryStrip is the acidic end and should point toward the anode (+).

Detection method was notsensitive enough.

Use another detection method (e.g. silver staining instead of Coomassie blue staining).

Individual proteins appear as multiplespots or are missing, unclear, or in wrong position

Protein carbamylation. Do not heat any solutions containing urea above 30 ºC, as cyanate, a urea degradation the product, will carbamylate proteins, changingtheir pI.

Vertical gap in 2-D pattern

Impurities in sample. Modify sample preparationImpurities in rehydration solution components.

Use only high-quality reagents. Deionize urea solutions.

Troubleshooting of 2 D Gel Electrophoresis

Symptom Possible cause Remedy

Vertical gap in 2-D pattern

Bubble between DryStrip gel and top surface of second-dimension gel.

Ensure that no bubbles are trapped between the DryStrip gel and the top surface of the second-dimension gel.

Urea crystals on the surface of the DryStrip gel.

Allow residual equilibration solution to drain from the DryStrip gel before placing the strip on the second-dimension gel.

Horizontal stripes across gel

Impurities in agarose overlayor equilibration solution.

Prepare fresh agarose overlay and equilibration solution

Disadvantages of 2D Gel Electrophoresis

• This technique include a large amount of sample handling, • Limited reproducibility, and a smaller dynamic range than some

other separation methods. • Difficulty in separation of hydrophobic proteins (Corthals et al,

2000; Wilkins et al, 1998)• It is also not automated for high throughput analysis. • Certain proteins are difficult for 2D-PAGE to separate, including

those that are in low abundance, acidic, basic, very large, or very small.

• Separation of proteins by two-dimensional gel electrophoresis (2-DE) coupled with identification of proteins through peptide mass fingerprinting (PMF) by MALDI-TOF MS is the widely used technique for proteomic analysis.

• In this work, they investigated the reliability of using raw genome sequences for identifying proteins. The method is demonstrated for proteomic analysis of Klebsiella pneumoniae grown anaerobically on glycerol.

• For 197 spots excised from 2-DE gels and submitted for mass spectrometric analysis 164 spots were clearly identified as 122 individual proteins.

• 95% of the 164 spots can be successfully identified merely by using peptide mass fingerprints and a strain-specific protein database (ProtKpn) constructed from the raw genome sequences of K. pneumoniae. Cross-species protein searching in the public databases mainly resulted in the identification of 57% of the 66 high expressed protein spots in comparison to 97% by using the ProtKpn database.

• In conclusion, the use of strain-specific protein database constructed from raw genome sequences makes it possible to reliably identify most of the proteins from 2-DE analysis simply through peptide mass fingerprinting.

• The serum proteins were separated by two-dimensional electrophoresis (2-DE); 29 different gene products were identified.

• Proteins represented by 25 spots/spot groups were identified by tandem nanoelectrospray mass spectrometry (MS), four by matrix-assisted laser desorption ionization time-of-flight (TOF) MS and one was sequenced by TOF-TOF technology.

• The identities of four proteins were deduced by similarity to the human plasma protein database.

• In selected cases, i.e. the immunoglobulins, immunoblotting with specific antibodies provided additional information about the respective proteins.

• Albumin was detected as the full-length protein and as fragments of various sizes. Spots representing products of different mass and charge were also detected for α1-antitrypsin, haptoglobin and transthyretin.

• They are able to identify almost all moderate to high abundance proteins stained in the serum 2-DE pattern.

• Acute phase proteins (APP) have been identified in whey and sera from healthy and mastitis cows through the proteomic analysis using two-dimensional electrophoresis (2-DE) coupled with Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS).

• Although normal and mastitis serum samples show relatively similar protein composition, marked differences in expression levels and patterns can be observed.

• Conversely, normal and mastitis whey showed a very different composition, likely due to extravasation of blood proteins to the mammary gland.

• Different isoforms from the most abundant protein in milk, casein, were detected in both normal and mastitis whey. Other proteins, such as lactotransferrin, were only detected in the inflamed animal samples.

• Immunoglobulins showed different patterns but not increased levels in the inflamed whey.• Also, many cellular proteins in mastitis cow's whey, that were absent from healthy cow's

milk. They are responsible for the great change in composition between normal and mastitis whey, especially those which exert a biological function related to immune defense.

• Data collected in this work are of interest for gaining information about physiological changes in protein patterns in different fluids and, the correspondent modifications as result of an acute phase process in farm.

• The serum proteome may be a good tool to identify useful protein biomarkers for recognising sub-clinical conditions and overt disease in sheep.

• In order to characterize normal protein patterns and improve knowledge of molecular species-specific characteristics, they generated a two-dimensional reference map of sheep serum.

• The possible application of this approach was tested by analysing serum protein patterns in ewes with mild broncho-pulmonary disease, which is very common in sheep and in the peripartum period which is a stressful time, with a high incidence of infectious and parasitic diseases. They found overall, 250 protein spots were analyzed, and 138 identified.

• Compared with healthy sheep, serum protein profiles of animals with rhino-tracheo-bronchitis showed a significant decrease in protein spots identified as transthyretin, apolipoprotein A1 and a significant increase in spots identified as haptoglobin, endopin 1b and alpha1B glycoprotein.

• In the peripartum period, haptoglobin, alpha-1-acid glycoprotein, apolipoprotein A1 levels rose, while transthyretin content dropped.

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Further readings

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