Identification of Proteins from Two-Dimensional Electrophoresis Gels

Chapter 11

Identification of Proteins from TwoDimensional Electrophoresis Gels by Peptide Mass Fingerprinting Downloaded by STANFORD UNIV GREEN LIBR on October 14, 2012 | http://pubs.acs.org Publication Date: May 5, 1996 | doi: 10.1021/bk-1995-0619.ch011

David P. Arnott, William J. Henzel, and John T. Stults Protein Chemistry Department, Genentech, Inc., 460 Point San Bruno Boulevard, South San Francisco, CA 94080

As part of a project to identify factors involved in congestive heart failure, differences in protein expression levels between normal and enlarged (hypertrophic) heart cells were identified. In initial experiments, two-dimensional gel electrophoresis was used to separate the proteins from normal neonatal rat cardiac myocytes. The proteins were electroblotted to a membrane and identified by staining. Proteins of interest were cleaved into peptides with an in situ enzymatic digestion method. The masses of the peptides were determined by capillary high performance liquid chromatography electrospray ionization mass spectro metry and, when possible, partial sequences were obtained by subsequent liquid chromatography tandem mass spectrometry (LC-MS/MS) experi ments. These data were used to search a protein sequence database with the program FRAGFIT. The program theoretically cleaves each protein in the database. By comparison of the experimentally-determined peptide masses and the theoretical masses, the program identifies the protein if it exists in the database. A partial sequence from the LC-MS/MS experiment was used to increase the specificity of the search. With this approach, eight proteins present in low picomole quantities on 2-D gels from cardiac myocytes have been identified, with 100 fmol or less of each peptide component required for the mass spectral experiments. Methods for the determination of a protein's amino acid sequence have evolved considerably during the past forty years. Amino acid sequencing by Edman degradation was the first generation method (i). This method, now extensively automated and refined, is still widely used today, though rarely for the sequence determination of an entire protein. Methods for gene cloning and nucleotide sequencing, developed in the 1970's (2), made it generally much easier to infer the primary structure from the cDNA sequence. This second generation method is now the one most commonly used. Nonetheless, the determination of partial amino acid sequence by automated Edman degradation, from which oligonucleotide probes are designed, is typically a prerequisite for the cloning process. The need to determine novel protein sequences is rapidly being supplanted by the need to identify proteins from a database. The explosive growth of protein sequence databases has significantly raised the likelihood that the sequence of a 0097-6156/95Λ)619-0226$12.00Λ) © 1996 American Chemical Society

In Biochemical and Biotechnological Applications of Electrospray Ionization Mass Spectrometry; Snyder, A.; ACS Symposium Series; American Chemical Society: Washington, DC, 1996.

Downloaded by STANFORD UNIV GREEN LIBR on October 14, 2012 | http://pubs.acs.org Publication Date: May 5, 1996 | doi: 10.1021/bk-1995-0619.ch011

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Identification of Proteins from Electrophoresis Gels

protein of interest is already known. Furthermore, recent strategies for generating partial sequences of cDNA's (expressed sequence tags - EST's) (3, 4) should lead to databases that contain partial nucleotide sequences for virtually all human proteins within the next 1-2 years. Finally, the human genome project will, upon its completion, produce the entire sequence for each human gene. Recent proposals suggest that completion of the human genome sequence (5) could be completed as early as 2001 (6). Sequencing of other genomes is also progressing rapidly. Although database searching with Edman-generated data is commonplace, a new method, now in its infancy, could potentially become the common method for protein identification. This method no longer involves Edman degradation, but is a liquid chromatography-mass spectrometry-based method (LC-MS/MS) for database searching. This next-generation method has become known as peptide mass fingerprinting, and it is shown conceptually in Figure 1. Peptides are generated enzymatically or chemically from a protein of interest. The peptide masses are determined by mass spectrometry. These masses are used as input to a computer program that theoretically digests each protein in a sequence database according to the specificity of the cleavage reaction used. The experimental peptide set is matched with the peptide set for each protein in the database to determine the identity of the protein, if it exists in the database. As few as 3-4 peptide masses may be sufficient to determine a protein identity, with more masses serving to increase the confidence in the match. This method was originally described by Henzel et al. in 1989 (7), and its utility subsequently demonstrated by our group (8-10) and others (11-16). The method has been rapidly adopted (see reviews (77, 18) ) and used successfully by a growing number of other research groups (19-23). Many data systems sold with commercial mass spectrometers now include software for database matching. Enhancements to the technique include the use of partial peptide sequence (24) and high resolution mass measurements (25). A particularly innovative approach uses a protein sequence database to generate theoretical MS/MS fragment ion spectra, based on a matching precursor mass, to match with the authentic MS/MS spectrum (26, 27). An important application of peptide mass fingerprinting is the identification of proteins separated by two-dimensional (2-D) electrophoresis. 2-D electrophoresis is an extremely high resolution method of protein separation (28), capable of resolving more than 2000 proteins in a single gel (29). Comparisons of 2-D gel images have shown differences in protein expression levels between normal and diseased tissue, and altered expression levels in transformed cells and cells treated with a variety of factors (30). Identification of proteins on the gels is a difficult task, due to the large numbers of proteins, and the vanishingly small amounts of protein found in a typical spot on a single 2-D gel (approx. 10 pmol for the most abundant proteins on analytical gels). The number of proteins of interest that must be identified can be reduced substantially by comparing 2-D gel images to identify those spots of interest (31). Furthermore, the availability of a number of 2-D gel databases (32-35), some accessible over the World Wide Web (36, 37), can help to identify some of the spots. The use of these databases, however, requires that the same electrophoresis protocol be duplicated precisely and that the same tissue or cell line be used. These constraints limit the widespread usage of 2-D electrophoresis databases based solely upon image matching. Due to its speed and sensitivity, peptide mass fingerprinting has been used successfully to identify proteins from 2-D gels (8, 19-23). One of the keys to this approach is the efficient generation of peptides from the protein spots. Two methods have been used successfully. A protein spot in a gel, following staining and excision of the spot, can be digested in the gel (38-40). The peptides are extracted from the gel matrix for subsequent measurement. Alternatively, the proteins can be electroblotted to a membrane where they are subsequently stained. A spot of interest


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Protein Sequence Database

Peptide Mixture

LC-MS LC-MS/MS

"digest"

Theoretical Peptide Masses

Peptide Masses (partial sequence)

FRAGFIT program

Protein Match

Figure 1. Schematic diagram of peptide mass fingerprinting method. The peptides are generated by in situ reduction/alkylation/digestions of electroblotted proteins. Masses for observed peptides are matched against theoretically digested proteins from a database. If the protein is present in the database, a match will be found if the conditions for matching are satisfied (see text).



11. ARNOTT ET AL.


is cut from the membrane and an in situ digest is performed directly on the membrane (41-45). The peptides are analyzed after extractionfromthe membrane. In situ digestion on a membrane is favored because fewer auto-proteolytic peptides are observed (20). In our earlier results we showed that proteins from a single 2-D gel of an E. coli lysate could be identified by mass alone (8). Mass measurement was by matrixassisted laser desoφtion/ionization-time-of-flight mass spectrometry (MALDI-TOF). As few as four peptide masses were sufficient to uniquely identify a protein, with as little as 20 fmol of a protein digest needed for the mass measurement. The results were confirmed by conventional automated Edman degradation. We demonstrate the use of peptide mass fingerprinting for the identification of proteins from 2-D gels of lysates from neonatal rat cardiac myocytes. This work is the first step in a project to identify proteins that are involved in cardiac hypertrophy, one of the major contributors to congestive heart failure. Peptide mass measurement and partial peptide sequence were obtained by LC-MS and LC-MS/MS, respectively, utilizing electrospray ionization (ESI). Experimental Materials. All reagents were of the highest quality available. All organic solvents were HPLC grade. Water was purified with a Milli-Q system (Millipore). Two-dimensional Gel Electrophoresis. The cell pellet derived from a single plate of cultured neonatal rat cardiomyocytes was dissolved in 10 lysis buffer (8 M urea, 2% Pharmalyte 3-10 ampholytes, 2% Triton X-100, 2% 2-mercaptoethanol, 0.1 mM 4-(2-aminoethyl)-benzenesulfonylfluoride (AEBSF) protease inhibitor). Insoluble material was removed by centrifugation at 4°C for 45 min in an Eppendorf centrifuge. Two or three such samples were pooled and diluted 2:1 with sample solution (6M urea, 2% Pharmalyte 3-10 ampholytes, 0.5% Triton X-100, 2% 2-mercaptoethanol) yielding a total protein content of approximately 400 μg. Two-dimensional electrophoresis was carried out using a Pharmacia Multiphor II system. First dimension isoelectric focusing (IEF) was performed with immobilized pH gradient strips (pH 3-10, length 11 cm) rehydrated in a solution containing 6M urea, 0.5% Triton X-100, 10 mM DTT, 2 mM acetic acid. The immobilized pH gradient (IPG) strips were placed in the immobiline strip tray according to the manufacturer's instructions and samples were loaded at the acidic end of each strip. Isoelectric focusing was carried out at 15°C over 16 hours: 300 V for 3 h; 300-2000 V over 5 h; 2000 V for 8 h. The strips were equilibrated for 20 min in SDS buffer (50 mM Tris HC1 pH 6.8, 6M urea, 30% glycerol, 1% w/v SDS, 0.005% Bromophenol Blue) and applied (two can be run simultaneously) to Pharmacia SDS-PAGE gels (245 χ 180 χ 0.5 mm, 12-14 % polyacrylamide gradient, pre-cast on a plastic support film). Electrophoresis proceeded at 20 mA constant current until the dye front migrated 5 mm into the gel, at which point the IPG strip was removed. Electrophoresis then continued at 40 mA constant current until the dyefrontreached the end of the gel. Proteins were electroblotted onto poly(vinylidene)difluoride (PVDF) mem branes (Immobilon-P Q, Millipore) at 250 mA constant current in 10 mM CAPS buffer, pH 11, for one hour. The blots were stained with Coomassie Brilliant Blue R-250 (0.1% w/v in 50% methanol) for 1 min, and destained with a solution of 50% methanol, 10% acetic acid until protein spots became visible. The membranes were rinsed thoroughly with deionized water, and allowed to air dry. s

In Situ Digestion. Protein spots of interest were cut from the blot, destained, reduced, alkylated, and digested with endoproteinase Lys-C (8). The excised spots


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(0-1000p.s.i)

Figure 2. Capillary HPLC-ESI interface. A capillary HPLC column (75-100 μπι) is inserted directly into the tip of the ESI needle assembly. The sample is loaded by pressure in a stainless steel "bomb," as shown, with the end of the column placed in the Eppendorf tube that contains the protein digest. Following loading, the capillary column is removed from the bomb and connected via a splitter to the HPLC pump. In this way, an injection loop is avoided, and low flow rates (approx. 0.2 μΐ/min) are achieved with a conventional HPLC pump. The electrospray stability is enhanced by the use of a 70% methanol/0.1 M acetic acid liquid sheath flowing at 1.2 μΐ/min.



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were transferred to siliconized 0.65 mL microcentrifuge tubes and wetted with 2 pL methanol. Residual stain was removed by the addition of 50 uJL deionized water and 200 methanol, with vortexing for one minute, followed by addition of 50 μι. chloroform and further vortexing. The solvent was then removed by pipette. Destaining, although not essential, was found to reduce interferences in the subsequent mass spectra. Reduction of disulfide bonds was accomplished by addition of 100 pL reduction buffer (10 mM DTT, 5 mM EDTA, 500 mM Tris HC1) to the wet membrane, with incubation at 45°C for one hour. Cysteine residues were then carboxymethylated by addition of 10 pL 200 mM iodoacetic acid in 0.5 M NaOH, followed by incubation in the dark for 20 min. The membranes were washed three times with 10% acetonitrile, and vortexed gently for 20 min in a solution of 0.5% w/v PVP-360,0.1% acetic acid. The solution was removed and the membranes washed twice with 10% acetonitrile, once with 20% acetonitrile, and once with digestion buffer (50 mM ammonium bicarbonate, 10% acetonitrile). Proteins were digested overnight at 37 °C with 0.05 μg endoproteinase Lys-C (Wako) in 15 pL digestion buffer. The membrane was removed from the digestion buffer, washed once with 5 pL of fresh digestion buffer, which was then pooled with the remaining buffer, acidified with 1 pL glacial acetic acid, and reduced in volume to

Identification of Proteins from Two-Dimensional Electrophoresis Gels

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