Molecular Diagnosis of Infectious Diseases

443 Pages·2001·6.773 MB·English

by  Roland Kontermann, Stefan Dübel

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Proteomic Approaches to Antigen Discovery 3
1
Proteomic Approaches to Antigen Discovery
Karen M. Dobos, John S. Spencer, Ian M. Orme, and John T. Belisle
Abstract
Proteomics has been widely applied to develop two-dimensional polyacrylamide
gel electrophoresis maps and databases, evaluate gene expression profiles under dif-
ferent environmental conditions, assess global changes associated with specific muta-
tions, and define drug targets of bacterial pathogens. When coupled to immunological
assays, proteomics may also be used to identify B-cell and T-cell antigens within com-
plex protein mixtures. This chapter describes the proteomic approaches developed by
our laboratories to accelerate the antigen discovery program for Mycobacterium
tuberculosis. As presented or with minor modifications, these techniques may be uni-
versally applied to other bacterial pathogens or used to identify bacterial proteins pos-
sessing other immunological properties.
Key Words: Proteomics; antigen; B-cell; T-cell; Mycobacterium; bacteria; pathogens.
1. Introduction
The ability to identify antigens produced by bacterial pathogens that are effective
diagnostic or vaccine candidates of disease depends on multiple variables. Two of the
most important variables are the source of clinical specimens (immune T-cells or sera)
used to identify potential antigens and the source or nature of the crude materials
containing the putative antigens. Factors that influence the usefulness of clinical speci-
mens include whether samples were obtained from diseased individuals or experimen-
tally infected animals and the state of disease at the time of specimen collection.
Likewise, the choice between native bacterial products and recombinant products as
the starting material for antigen discovery efforts may significantly influence whether
a useful antigen will be identified. An equally important factor is the number of poten-
tial antigens that can be screened in a single experiment. The use of recombinant
molecular biology methods and the screening of large recombinant libraries is one
approach toward maximizing the number of potential antigen targets (1–4). Although
recombinant expression systems have been widely used for antigen identification, there
From: Methods in Molecular Medicine, vol. 94: Molecular Diagnosis of Infectious Diseases, 2/e
Edited by: J. Decker and U. Reischl © Humana Press Inc., Totowa, NJ
3
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4 Dobos et al.
are several potential drawbacks. Specifically, variability between the folding of a
recombinant and native protein can complicate B-cell antigen discovery efforts
(5), and the contamination of recombinant proteins with other bacterial products
such as endotoxin is a major obstacle for cellular assays used to identify T-cell
antigens (6).
The ability to sequence whole genomes rapidly and the availability of several
fully annotated bacterial genomes have profoundly altered the basic experimental
approach to the study of bacterial physiology and pathogenesis (7,8). Previous to the
sequencing of whole bacterial genomes, investigators would typically focus on a
relatively small number of genes or gene products and develop specific assays to
assess the activities or relevance of these gene products. In contrast, the availability
of whole genome sequences has now allowed for the development of methodologies
such as DNA microarrays and proteomics to identify all the genes that are poten-
tially involved in a specific cellular process (9–11). Unlike DNA microarrays, the
technologies commonly used for proteomics studies [two-dimensional polyacryla-
mide gel electrophoresis (2D-PAGE) and mass spectrometry (MS) of peptides] have
been around for decades (12,13). The power of these two technologies was brought
together by the need to assess rapidly all the proteins produced in a particular bacte-
rial species, as well as the development of innovative software that allows for the
interrogation of MS data against genome sequences to identify proteins of interest
(14). These technologies and the philosophy that we no longer need to focus on
select sets of proteins, but should be evaluating the complete proteome in a single
experiment can now be applied to antigen discovery efforts. Moreover, proteomics
technologies allow antigen discovery programs to focus on large sets of native pro-
teins and eliminate a reliance on recombinant technologies to expand the pool of
proteins to be screened.
In our laboratory, 2D-PAGE, Western blot analysis, and liquid chromatography-
mass spectrometry (LC-MS) were used to define 26 proteins of Mycobacterium tuber-
culosis that reacted with patient sera; three of these subsequently were determined to
have significant potential as serodiagnostic reagents (15,16). This approach is a
relatively facile method to screen for B-cell antigens. The use of proteins resolved by
2D-PAGE and transferred to nitrocellulose was also applied to T-cell antigen identifi-
cation (17). Although this work revealed several potential T-cell antigens, there are
restrictions to its use. In particular, the concentration of protein tested is unknown and
the amount of protein obtained is most likely insufficient for multiple assays. Thus, to
increase the protein yield, we recently applied 2D liquid-phase electrophoresis (LPE)
coupled with an in vitro interferon-γ (IFN-γ) assay and LC-tandem MS to identify
30 proteins from the culture filtrate and cytosol of M. tuberculosis that possess a potent
capacity to induce antigen-specific IFN-γ secretion from the splenocytes of M. tuber-
culosis-infected mice (18). In this chapter, we detail the proteomics approach used in
the identification of candidate B- and T-cell antigens from M. tuberculosis. However,
these methods can be universally applied to the discovery of antigens from other
bacterial pathogens as well as parasites.
01/Dobos/1-18/F 4 09/26/2003, 1:59 PM
Proteomic Approaches to Antigen Discovery 5
2. Materials
2.1. Preparation of Subcellular Fractions
1. Bacterial cell cultures (400 mL or greater) (see Note 1).
2. Breaking buffer: phosphate-buffered saline (PBS; pH 7.4), 1 mM EDTA, 0.7 µg/mL
pepstatin, 0.5 µg/mL leupeptin, 0.2 mM phenylmethylsulfonyl fluoride (PMSF), 0.6 µg/mL
DNase, µg/mL RNase (see Note 2).
3. NaN .
3
4. Dialysis buffer: 10 mM ammonium bicarbonate, 1 mM dithiothreitol (DTT), 0.02% NaN .
3
5. 10 mM ammonium bicarbonate.
6. Vacuum pump.
7. Amicon ultrafiltration unit with a 10,000 Da MWCO membrane (Millipore, Bedford,
MA; cat. no. PLGC07610).
8. 0.2 µm Zap Cap S Plus bottle filtration units (Nalgene, Rochester, NY).
9. Dialysis tubing (3500 Da MWCO).
10. French Press and French Press cell.
11. Sterile 250-mL high-speed centrifuge tubes.
12. Sterile 30-mL ultracentrifuge tubes.
2.2. Identification of B-Cell Antigens
via 2D Western Blot Analysis with Protein/Antigen Double Staining
2.2.1. Optimization of Serum Titers for Detection of Antigens by Western Blot
1. Human or experimental animal sera samples (see Notes 3 and 4).
2. 15% sodium dodecyl sulfate (SDS)-PAGE gels (7 × 10 cm).
3. Protein molecular weight standards.
4. Laemmli sample buffer (5X): 0.36 g Tris-base, 5.0 mL glycerol, 1.0 g SDS, 5.0 mg bro-
mophenol blue, 1.0 mL β-mercaptoethanol; QS to 10 mL with Milli-Q water, vortex, and
store at 4°C or less (19).
5. SDS-PAGE running buffer: 3.02 g Tris-base (pH 8.3), 14.42 g glycine, 1 g SDS per 1 L.
6. Nitrocellulose membrane, 0.22 µm.
7. Transfer buffer: 3.03 g Tris-base (pH 8.3), 14.4 g glycine (pH 8,3), 800 mL H O, 200 mL
2
CH OH. Dissolve reagents in water before adding CH OH (20).
3 3
8. Tris-buffered saline (TBS): 50 mM Tris-HCl (pH 7.4), 150 mM NaCl.
9. Wash buffer: TBS containing 0.5% vol/vol Tween 80.
10. Blocking buffer: wash buffer containing 3% w/v bovine serum albumin (BSA).
11. Anti-human IgG conjugated to horseradish peroxidase (HRP).
12. BM Blue POD substrate, precipitating (Roche Molecular Biochemicals, Indianapolis, IN;
cat. no. 1442066).
13. Mini incubation trays for 2–4 mm nitrocellulose membrane strips (Bio-Rad, Hercules,
CA; cat. no. 170-3902).
2.2.2. 2D Western Blot Analysis for Identification of B-Cell Antigens
1. Isoelectric focusing (IEF) rehydration buffer: 8 M urea, 1% 3[(3-cholamidopropyl)
dimethylammonio]-1-propanesulfonate (CHAPS), 20 mM DTT, 0.5% ampholytes,
0.001% bromphenol blue (see Notes 5–7).
2. Immobilized pH gradient (IPG) strips (21) (see Note 8).
3. SDS-PAGE equilibration buffer: 150 mM Tris-HCl (pH 8.5), 0.2% SDS, 10% glycerol,
20 mM DTT, and 0.001% bromphenol blue.
01/Dobos/1-18/F 5 09/26/2003, 1:59 PM
6 Dobos et al.
4. 1% agarose dissolved in Milli-Q water.
5. Preparative SDS-PAGE gels (16 × 20 cm).
6. Protein molecular weight standards.
7. SDS-PAGE running buffer (see Subheading 2.2.1., item 5).
8. Coomassie stain: 1% coomassie brilliant blue R-250 in 40% methanol, 10% acetic acid
(see Note 9).
9. Coomassie destain 1: 40% methanol, 10% acetic acid.
10. Coomassie destain 2: 5% methanol.
11. Transfer buffer (see Subheading 2.2.1., item 7).
12. Nitrocellulose, 0.22 µm.
13. Digoxigenin-3-O-methylcarbonyl-ε-aminocaproic acid-N-hydroxysuccinimide ester
(DIG-NHS) (Roche Molecular Biochemicals; cat. no. 1333054); 0.5 mg/mL in N,N-
dimethylformamide (DMF).
14. 50 mM potassium phosphate buffer (pH 8.5).
15. TBS: 50 mM Tris-HCl (pH 7.4), 150 mM NaCl.
16. Nonidet P-40, 10% solution.
17. H O , 30% solution.
2 2
18. Anti-digoxigenin-alkaline phosphatase, Fab fragments (Roche Molecular Biochemicals;
cat. no. 1093274).
19. INT/BCIP stock solution (Roche Molecular Biochemicals; cat. no. 1681460).
20. INT/BCIP buffer: 100 mM Tris-HCl (pH 9.5), 50 mM MgCl , 10 mM NaCl.
2
21. Anti-human IgG conjugated to HRP.
22. IPGphor IEF unit (Amersham Biosciences, Piscataway, NJ) or similar system.
23. SDS-PAGE electrophoresis unit.
24. Gel documentation system.
25. PDQuest 2D-gel analysis software (Bio-Rad) or similar software.
2.2.3. Molecular Identification of Serum Reactive Proteins
1. 0.2 M ammonium bicarbonate.
2. Trifluoroacetic acid (TFA; 10% solution).
3. Destain solution: 60% acetonitrile, in 0.2 M ammonium bicarbonate.
4. Extraction solution: 60% acetonitrile, 0.1% TFA.
5. Modified trypsin, sequencing grade (Roche Molecular Biochemicals; cat. no. 1418025).
6. Washed microcentrifuge tubes (0.65 mL) (see Note 10).
7. Electrospray tandem mass spectrometer (such as LCQ classic, Thermo-Finnigan) coupled
to a capillary high-performance liquid chromatography (HPLC) device.
8. Capillary C -reverse phase (RP)-HPLC column.
18
9. Sequest software (14) for interrogating MS and MS/MS data against genomic or protein
databases, or similar software.
2.3. Identification of T-Cell Antigens
2.3.1. 2D-LPE of Subcellular Fractions
1. IEF protein solubilization buffer: 8 M urea, 1 mM DTT, 5% glycerol, 2% Nonidet P-40,
and 2% ampholytes (pH 3.0–10.0 and pH 4.0–6.5 in a ratio of 1:4; see Note 7).
2. Preparative SDS-PAGE gels (16 × 20 cm).
3. SDS-PAGE running buffer (see Subheading 2.2.1., item 5).
4. Laemmli sample buffer (see Subheading 2.2.1., item 4).
5. 10 mM ammonium bicarbonate.
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Proteomic Approaches to Antigen Discovery 7
6. Rotofor preparative IEF unit (Bio-Rad).
7. Whole Gel Eluter (Bio-Rad).
2.3.2. Assay of IFN-γ Induction for Identification of T-Cell Antigens
1. Spleens from infected and naive mice (see Note 11).
2. Complete RPMI medium (RPMI-1640 medium with L-glutamine, supplemented with
10% bovine fetal calf serum and 50 µM β-mercaptoethanol) (see Note 12).
3. Hanks’ balanced salt solution (HBSS).
4. Gey’s hypotonic red blood cell lysis solution: 155 mM NH Cl, 10 mM KHCO ; use pyro-
4 3
gen-free water and filter-sterilize.
5. IFN-γ enzyme-linked immunosorbent assay (ELISA) assay kit (Genzyme Diagnostics,
Cambridge, MA) (see Note 13).
6. Concanavalin A (Con A) (see Note 14).
7. 70-µm nylon screen (Becton/Dickinson, Franklin Lakes, NJ; cat. no. 35-2350).
8. 96-well sterile tissue culture plates with lids.
9. 96-well microtiter ELISA plates, Dynex Immunlon 4 (Dynex, Chantilly, VA).
10. Conical polypropylene centrifuge tubes (15 and 50 mL).
11. Sterile tissue culture grade Petri dishes (60 mm).
12. Syringes (3 mL) with 1-inch 22-gage needles.
13. Hemocytometer.
14. Tissue culture incubator (5% CO , 37°C).
2
15. ELISA plate reader.
3. Methods
3.1. Preparation of Bacterial Subcellular Fractions (see Note 15)
1. Grow bacterial cells to mid log phase. The medium used should be devoid of exogenous
proteins, such as BSA, that may interfere with 2D-PAGE analyses (see Note 1).
2. Harvest cells by centrifugation at 3500g. Decant the culture supernatant and save.
Wash the cell pellet with PBS (pH 7.4) and freeze until preparation of subcellular frac-
tions (see Note 16).
3. Add NaN to the culture supernatant at a final concentration of 0.04% (w/v).
3
4. Filter the supernatant using a 0.2-µm filtration unit.
5. Concentrate the culture filtrate to 0.5% of its original volume using an Amicon ultrafiltra-
tion unit fitted with a 10-kDa MWCO membrane.
6. Dialyze the concentrated culture filtrate proteins (CFP) against dialysis buffer with at
least two changes of this buffer, followed by a final dialysis step against 10 mM ammo-
nium bicarbonate.
7. Filter-sterilize the dialyzed CFP using a 0.2-µm syringe filter or filtration unit.
8. Determine the protein concentration.
9. Store the final culture filtrate preparation at –80°C (see Note 17).
10. Place the frozen cell pellet at 4°C and thaw.
11. Suspend the cells in ice-cold breaking buffer to a final concentration of 2 g of cells
(wet weight) per mL of buffer.
12. Place the cell suspension in a French press cell and lyse via mechanical shearing by
applying 20,000 psi of pressure with a French press. Collect the lysate from the French
press cell and place on ice (see Note 18).
13. To the lysate add an equal volume of breaking buffer and mix.
01/Dobos/1-18/F 7 09/26/2003, 1:59 PM
8 Dobos et al.
14. Remove unbroken cells by centrifugation of the lysate at 3500g, 4°C in the tabletop cen-
trifuge for 15 min.
15. Collect the supernatant; this is the whole cell lysate.
16. Further separation of the whole cell lysate into cell wall, cell membrane, and cytosol is
achieved by differential centrifugation (22). First, centrifuge the lysate at 27,000g, 4°C
for 30 min. Collect the supernatant and again centrifuge under the same conditions.
Collect the supernatant and store at 4°C.
17. Suspend the 27,000g pellets in breaking buffer, combine them, and centrifuge at 27,000g,
4°C for 30 min. Discard the supernatant, and wash the pellet twice in breaking buffer.
The final 27,000g pellet represents the purified cell wall. Suspend this pellet in 10 mM
ammonium bicarbonate and extensively dialyze against 10 mM ammonium bicarbonate.
18. Store the dialyzed cell wall suspension at –80°C, after estimating the protein concentra-
tion (see Note 17).
19. Add the supernatant collected in step 16 to ultracentrifuge tubes and centrifuge at
100,000g, 4°C for 4 h.
20. Collect the supernatant and centrifuge again under the same conditions.
21. The final supernatant solution represents the cytosol fraction. Dialyze the cytosol exten-
sively against 10 mM ammonium bicarbonate, determine the protein concentration, and
store at –80°C (see Note 17).
22. Suspend the 100,000g pellets obtained in steps 19 and 20 in breaking buffer, combine,
and again centrifuge at 100,000g, 4°C for 4 h. This should be repeated twice.
23. The final 100,000g pellet represents the total membrane. Suspend the membranes in
10 mM ammonium bicarbonate, dialyze extensively against 10 mM ammonium bicar-
bonate, determine the protein concentration, and store at –80°C (see Note 17).
3.2. Identification of B-Cell Antigens
via 2D Western Blot Analysis with Protein/Antigen Double Staining
3.2.1. Optimization of Serum Titers for Detection of Antigens by Western Blot
1. Obtain and thaw a 100-µg aliquot (based on protein concentration) of the subcellular
fraction to be analyzed. One 100-µg aliquot will provide enough protein to optimize one
patient’s serum or one sera pool and one matched control.
2. Dry the samples using a lyophilizer or speed vac.
3. Suspend the subcellular fraction in 80 µL PBS (pH 7.4).
4. Add 20 µL of 5X Laemmli sample buffer to the sample and heat at 100°C for 5 min.
5. Apply 100 µL of sample to one preparative 15% SDS-polyacrylamide gel.
6. Resolve the proteins by 1D SDS-PAGE (19).
7. After the electrophoresis is completed, assemble the gel into a Western blot apparatus and
electrotransfer the proteins to a nitrocellulose membrane (20).
8. Remove the nitrocellulose membrane and cut 2-mm vertical strips.
9. Place individual nitrocellulose strips in the wells of a mini incubation tray. Add 500 µL of
blocking buffer to each well and rock for 1 h at room temperature (RT) or overnight at 4°C.
10. Thaw the serum samples and generate dilutions (200 µL for each dilution) of the sera
from 1:10 to 1:10,000 (see Note 19).
11. Incubate each strip with a single dilution of serum for 1 h at RT with gentle agitation.
12. Wash the strips repeatedly with 500 µL of wash buffer (minimally, five times).
13. Incubate the strips with 200 µL of the appropriate dilution of the anti-human IgG HRP for
1 h at RT with gentle agitation.
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Proteomic Approaches to Antigen Discovery 9
14. Wash the strips repeatedly with 500 µL of TBS (minimally, five times).
15. Develop the strips by addition of BM Blue POD substrate (200 µL, final volume per
strip).
16. Stop the reaction by decanting the substrate and rinsing the strips with Milli-Q water.
17. Determine optimum titer based on band intensity and background staining (see Note 20).
3.2.2. 2D Western Blot Analysis for Identification of B-Cell Antigens
1. Obtain and thaw 400-µg aliquots (see Note 17) of the subcellular fractions to be ana-
lyzed. For each subcellular fraction or analysis, at least two aliquots of the selected
subcellular fraction will be required, one for the 2D Western blot and one for a Coomassie-
stained 2D gel. More aliquots will be required if replicate Western blots are to be per-
formed or if comparisons between serum samples/pools are to be performed, such as a
comparison between infected and healthy control serum.
2. Dry each aliquot of the subcellular fraction by lyophilization (see Note 21).
3. Add 200 µL of rehydration buffer to each dried subcellular fraction and allow to stand at
RT for 4 h or at 4°C overnight. Gentle vortexing can be applied if required.
4. Centrifuge the samples at 10,000g for 30 min, and remove the supernatant without dis-
turbing the pellet (see Note 22).
5. Transfer samples to IPG strips and allow the strips to rehydrate per manufacturer’s rec-
ommendations.
6. Resolve proteins by IEF (12).
7. Remove strips from the IEF apparatus, and place in 16 × 150-mm glass test tubes with the
acidic end of the strip near the mouth of the tube. Apply 10 mL of SDS-PAGE equilibra-
tion buffer, and incubate at RT for 15 min.
8. Warm 1% agarose solution while IPG strips are equilibrating.
9. Assemble 16 × 20-cm preparative SDS-PAGE gels into an electrophoresis apparatus.
Add running buffer to cover the lower half of the gels and inner core of the electrophore-
sis apparatus.
10. Remove IPG strips, clip ends of IPG strips (where no gel is present), and guide each strip
into the well of the preparative SDS-PAGE gels using a flat spatula or small pipet tip.
The acidic end of the IPG strip should be next to the reference well for the molecular
weight standards.
11. Overlay each strip with 1% agarose. When adding agarose, move from one end of the
strip to the other. This helps prevent the trapping of air bubbles between the strip and
interface of the SDS-PAGE gel. Use enough agarose to cover the strip fully.
12. Add molecular weight standards to the reference well of each gel.
13. Resolve proteins in the second dimension by electrophoresis (19).
14. Remove the preparative SDS-PAGE gels. Stain one gel with Coomassie (see Note 9).
15. After staining, use a gel documentation system to scan a tif image of the gel. Ensure that
the gel is scanned under parameters compatible with the 2D gel analysis software that
will be used.
16. Place the gel in a storage tray, cover with Milli-Q water, seal, and store at 4°C.
17. Assemble the second gel into a Western blot apparatus and electrotransfer the proteins to
a nitrocellulose membrane (20).
18. Wash nitrocellulose membrane five times with 20 mL of 50 mM potassium phosphate
(pH 8.5).
19. Prepare total protein labeling solution by adding 10 µL of DIG-NHS and 20 µL of
10% Nonidet P-40 to 20 mL of 50 mM potassium phosphate buffer (pH 8.5).
01/Dobos/1-18/F 9 09/26/2003, 1:59 PM
10 Dobos et al.
20. Incubate membrane in labeling solution for 1 h at RT with gentle agitation.
21. Wash the membrane five times in 20 mL of TBS.
22. Incubate the labeled membrane in blocking buffer for 1 h at RT with gentle agitation.
23. Wash the membrane briefly with TBS.
24. Add serum at the titer optimized in Subheading 3.2.1. and incubate at RT for 1 h with
gentle agitation.
25. Wash the membrane five times with 20 mL TBS.
26. Add 20 µL of anti-digoxigenin-alkaline phosphatase to 20 mL of TBS and incubate the
membrane in this solution for 1 h with gentle agitation.
27. Wash the membrane five times with 20 mL of TBS.
28. Add anti-human IgG HRP diluted per manufacturer’s instructions into TBS, and incubate
the membrane in this solution for 1 h at RT with gentle agitation.
29. Wash the membrane five times with 20 mL of TBS.
30. Rinse the membrane briefly in Milli-Q water.
31. To visualize the serum-specific antigens, incubate the membrane in 10 mL of BM Blue
POD substrate without agitation. Watch the membranes for blue/purple color development.
32. Aspirate the substrate as soon as color develops, and rinse briefly with Milli-Q water.
33. Using a gel documentation system, capture a tif image of the Western blot showing the
serum reactive proteins.
34. To visualize the total protein profile on the Western blot, generate the alkaline phos-
phatase substrate by adding 75 µL of INT/BCIP stock solution to 10 mL of INT/BCIP
buffer. Add to the membrane, incubate without agitation, and watch for color development.
35. Aspirate the substrate solution when reddish brown spots are well defined, and rinse the
membrane briefly with Milli-Q water.
36. Using a gel documentation system, capture a tif image of the Western blot showing the
total protein profile.
37. Transfer the images of the Coomassie-stained gel, the serum reactive proteins, and the
total protein profile of the 2D Western blot to a 2D analysis program. Using this program,
match the spots of the three images to allow for identification of the protein spots within
the Coomassie-stained 2D gel that correspond to the serum reactive proteins.
3.2.3. Molecular Identification of Serum Reactive Proteins
1. From the Coomassie-stained 2D-gel, excise the protein spots corresponding to those
reactive to serum on the 2D Western blot.
2. Cut each gel slice into small pieces (1 × 1 mm), and place the gel pieces from each spot in
separate washed microcentrifuge tubes.
3. Destain by covering the gel pieces with destain solution, and incubate at 37°C for 30 min.
4. Discard the acetonitrile solution and repeat step 3 until the gel slices are completely
destained.
5. Dry the gel pieces under vacuum.
6. Dissolve 25 µg of modified trypsin in 300 µL of 0.2 M ammonium bicarbonate.
7. Add 3–5 µL of the trypsin solution to the gel slices.
8. Incubate at room temperature until the trypsin solution is completely absorbed by the gel,
approx 15 min.
9. Add 0.2 M ammonium bicarbonate in 10–15 µL increments to rehydrate the gel pieces
completely. Allow about 10 min for each aliquot of ammonium bicarbonate to be absorbed
by the gel. Also avoid adding an excess of ammonium bicarbonate solution.
10. Incubate the gel slices for 4–12 h at 37°C.
01/Dobos/1-18/F 10 09/26/2003, 1:59 PM
Proteomic Approaches to Antigen Discovery 11
11. Terminate the reaction by adding 0.1 vol of 10% TFA.
12. Collect the supernatant, and place it in a new washed microcentrifuge tube.
13. Add 100-µL of the extract solution to the gel slices and vortex.
14. Incubate the extract solution and gel slices at 37°C for 40 min.
15. Centrifuge the extract, collect the supernatant, and add it to the supernatant collected in
step 12.
16. Repeat steps 13–15.
17. Dry the extract under vacuum.
18. Store the dried peptide extracts at –20°C until analysis by LC-MS/MS (14).
3.3. Identification of T-Cell Antigens (see Note 23)
3.3.1. 2D-LPE of Subcellular Fractions
1. Obtain and thaw 250-mg aliquots of the subcellular fraction(s) to be analyzed.
2. Dry the subcellular fraction by lyophilization (see Note 21)
3. Solubilize the proteins by adding 60 mL IEF rehydration buffer, and incubate at RT for
4 h or at 4°C overnight.
4. Centrifuge the suspended material at 27,000g to remove particulates (see Note 22).
5. Collect the supernatant and apply this material to the Rotofor (Bio-Rad) apparatus per the
manufacturer’s instructions. Preparative IEF of the sample should be performed at a con-
stant power of 12 W until the voltage stops increasing and stabilizes (see Note 24).
6. Harvest the samples from the Rotofor per manufacturer’s instructions.
7. Evaluate 8–10 µL of each preparative IEF fraction by SDS-PAGE and Coomassie staining.
8. Pool those fractions that have a high degree of overlap in their protein profile as observed
by SDS-PAGE (see Note 25).
9. Dialyze the IEF fractions extensively against ammonium bicarbonate. After dialysis
determine the protein concentration.
10. Split the IEF fractions into 5-mg aliquots and dry by lyophilization or speed vac.
11. To separate each IEF fraction in the second dimension, solubilize 5 mg of each fraction in
1.6 mL of PBS and add 0.4 mL of 5X Laemmli sample buffer.
12. Apply each fraction to the preparative well of a 16 × 20-cm SDS-PAGE gel (see Note 26).
13. Resolve proteins in the second dimension by electrophoresis.
14. Remove polyacrylamide gels from glass plates and soak in 100 mL of 10 mM ammonium
bicarbonate for 30 min with one change of the 10 mM ammonium bicarbonate.
15. While the gel is equilibrating in 10 mM ammonium bicarbonate, assemble the Whole Gel
Eluter (Bio-Rad) per manufacturer’s instructions and fill the Whole Gel Eluter wells with
10 mM ammonium bicarbonate (see Note 27).
16. Cut SDS-PAGE gel to the dimension of the Whole Gel Eluter.
17. Lay the SDS-PAGE gel on top of Whole Gel Eluter wells. Orientate the gel so that protein
bands run parallel to the wells.
18. Complete the setup of the Whole Gel Eluter per manufacturer’s instructions, and elute the
proteins at 250 mA for 90 min.
19. At the end of the elution, reverse the current on the Whole Gel Eluter for 20 s.
20. Harvest the samples from the Whole Gel Eluter per manufacturer’s instructions, This will
yield 30 fractions of approximately 2.5 mL each.
21. Filter sterilize each fraction with a 0.2-µm PTFE syringe filter. (Use aseptic techniques
for subsequent manipulation of the 2D-LPE fractions.)
22. Determine the protein concentration of each fraction.
23. Split each fraction into 10-µg aliquots, lyophilize, and store at –80°C.
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