Proteomics Based on Peptide Fractionation by SDS-Free PAGE Yassel Ramos, Elain Gutierrez, Yoan Machado, Aniel Sa´nchez, Lila Castellanos-Serra, Luis J. Gonza´lez, Jorge Ferna´ndez-de-Cossio, Yasset Pe´rez-Riverol, La´zaro Betancourt, Jeovanis Gil, Gabriel Padro ´ n, and Vladimir Besada* Center for Genetic Engineering and Biotechnology, Apartado 6162, POB 10600, La Habana, Cuba Received December 11, 2007
Here we demonstrate the usefulness of peptide fractionation by SDS-free polyacrylamide gel electrophoresis and its applicability to proteomics studies. In the absence of SDS, the driving force for the electrophoretic migration toward the anode is supplied by negatively charged acidic aminoacid residues and other residues as phosphate, sulfate and sialic acid, while the resulting mobility depends on both the charge and the molecular mass of the peptides. A straightforward method was achieved for SDS-PAGE of proteins, enzyme digestion, peptide transfer and fractionation by SDS-free PAGE, which was named dual-fractionation polyacrylamide gel electrophoresis (DF-PAGE). This method increases the number of identified proteins 2.5-fold with respect to the proteins identified after direct analysis, and more than 80% of assigned peptides were found in unique SDS-free gel slices. A vast majority of identified peptides (93%) have pI values below 7.0, and 7% have pI values between 7.0 and 7.35. Peptide digests that were derived from complex protein mixtures were in consequence simplified as peptides that are positively charged are not recovered in the present conditions. The analysis of a membrane protein extract from Neisseria meningitidis by this approach allowed the identification of 97 proteins, including low-abundance components. Keywords: Fractionation • peptide • mass spectrometry • proteomic • protein • electrophoresis • SDS • PAGE • Neisseria meningitidis
1. Introduction Since 1970 when Laemmli1 introduced SDS as the main solubilizing agent for proteins, SDS-PAGE became the most important tool for protein characterization. This detergent homogenizes the negative charge density of the proteins allowing their separation by molecular size. Some years before, Ornstein2 had reported a pioneer work describing a discontinuous buffer system for negatively charged protein separation based on polyacrylamide gel electrophoresis (PAGE). In that procedure called originally “disc electrophoresis”, proteins and peptides are concentrated in a very thin starting zone previous to their separation into the resolving gel. This phenomenon is based on the “Kohlrausch function” that regulates the migration of the trailing and leading ions (glycinate and chloride, respectively, in the Ornstein system). Ions with electrophoretic mobility lower than the leading ion mobility and higher than the trailing ion mobility stay trapped into the boundary between these two ionic species. Once the boundary rises in the resolving gel, this stationary system becomes unstable at a pH value where the trailing ion is as fast as the leading ion. In these conditions, proteins and peptides migrate to the anode according to their electrophoretic mobility which is proportional to their charge and inversely proportional to their size. On the basis of this theory, several discontinuous buffer systems * To whom correspondence should be addressed. Phone, (573) 271-6022; e-mail,
[email protected]. 10.1021/pr700840y CCC: $40.75
2008 American Chemical Society
for electrophoresis have been developed3 and their application substantially improved the resolution of the gel-based protein separation techniques. Another key contribution to protein science was isoelectric focusing (IEF)4,5 that resolves proteins electrophoretically in a pH gradient up to the position where their net charge becomes zero. IEF and SDS-PAGE were further combined in tandem with great success, by a procedure known as bidimensional electrophoresis (2DE). This procedure allowed an impressive separation of thousands of proteins present in cell extracts.6 In the 1990s, mass spectrometry became the ideal tool for protein identification because of its sensitivity, high-throughput and its capability for primary structure elucidation, all of which started to be known as Proteomics. However, 2DE has several limitations for the analysis of hydrophobic proteins and basic proteins, and in addition, it is extremely laborious. In less than a decade, Proteomics has evolved and novel 2DE-free procedures have been developed, essentially based on the analysis by liquid chromatography coupled with mass spectrometry (LC-MS/MS)7 of complex peptide mixtures obtained after protein digestion. The number of identified proteins significantly increases when the method is applied to samples previously fractionated at the protein level using cell subfractionation by organelles,8 by solubility using different chaotropes and detergents,9,10 by chromatography11 or even by selective capture of peptides surrogates of each constituent protein.12 The introduction of multidimensional LC-MS/MS Journal of Proteome Research 2008, 7, 2427–2434 2427 Published on Web 04/15/2008
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Ramos et al.
Figure 1. Schematic representation of the strategy used for proteomic analysis (DF-PAGE). Proteins are separated according to their molecular mass in a SDS-miniPAGE. The unstained gel is cut into 16 × 3 mm slices, each slice is in-gel digested with trypsin, and the peptide mixture is transferred to a second SDS-free gel. Peptides are separated according to their charge/mass ratio. This gel is cut into 8 × 6 mm slices and peptides eluted from each slice are separated according to their hydrophobic properties in the LC-MS/MS analysis. The database interrogation is then performed using a composite data from the 8 LC-MS/MS experiments obtained for each protein fraction.
based approaches at the peptide level, in conjunction with stable isotope labeling allows high-throughput of protein identification and protein quantitation.7,13 The combination of different methods have been described for peptide separation, and strong cation exchange (SCX) and more recently isoelectric focusing (IEF), both coupled with reverse phase (RP) chromatography, are the techniques with the most resolving power. IEF is highly resolving for proteins and peptides, but a major drawback is keeping the solubility of the components during their focalization or once they are focalized as the use of potent solubilizing agents as SDS and guanidium chloride is incompatible with the technique and is ruled out. Two novel variants of IEF known as off-gel electrophoresis (OGE)14 and free-flow electrophoresis (FFE)15 have been applied to the separation of protein digests. Thousands of peptides are distributed according to their isoelectric point; their experimental pI constitutes an additional criterion for validating peptide and protein identification.16 More recently, a curious combination of IEF and SDS electrophoresis has been introduced. Basic immobilines are co-polymerized with acrylamide monomers creating a positively charged gradient in the gel matrix that allows the separation of protein-SDS micelles reaching steady-state conditions.17 An alternative to 2DE is the direct analysis of proteins in SDSPAGel bands by LC-MS/MS after proteolytic digestion.18 A complex protein sample is fractionated in a SDS gel, the unstained lane is cut into several segments, each segment is digested, and peptides are identified after LC-MS/MS. Around 20 proteins were identified per segment from 0.4 mg of a crude membrane preparation and the authors suggested that an additional peptide fractionation step could enhance the identification of low-abundance proteins comigrating with proteins present at higher levels. Considering the advantages of this approach for protein fractionation in presence of SDS, we developed a method that introduces a fractionation step at peptide level using a SDSfree PAGE. About 30 years ago, West et al. described the 2428
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separation of single protein digests at acidic or at basic pH in 40-50% polyacrylamide resolving gels in the absence of SDS.19 Nevertheless, in the 1980s, the procedure was abandoned in favor of the vast application of reversed phase chromatography that rapidly became the method of choice for peptide separation. Now we have reconsidered and modified this old procedure, demonstrating that efficient peptide fractionation can be accomplished in 15% PA gels, using standard discontinuous Tris-glycine buffer system in the absence of SDS (the original Ornstein system2). The method we now propose combines (1) fractionation of complex protein samples by SDS-PAGE in several bands that is followed by enzymatic digestion,18 (2) fractionation of peptide digests in SDS-free PAGE in conditions where a simpler surrogate is obtained as described in this paper, and (3) peptide separation and identification by conventional RP-LC MS/MS. The first separation benefits from the enhanced solubility of proteins in the presence of SDS. In the second step, peptides (derived from a defined molecular mass range of proteins) are fractionated along the electrophoretic run according to their charge-to-mass ratio. The high resolving power of this approach is due to the combination of three orthogonal principles of separation and allows the detection of peptide signals that cannot be discerned and analyzed when protein digests obtained from the first separation are directly analyzed by LCMS/MS. In consequence, there is a notable increase in the number of proteins identified in each gel band.
2. Materials and Methods 2.1. Materials and Reagents. Chemicals were of the highest quality from international suppliers. Recombinant streptokinase (rSK) was manufactured at the Centre for Genetic Engineering and Biotechnology (Havana, Cuba). Casein mixture was obtained from SIGMA (Saint Louis, MO). Modified porcine trypsin was from Promega (Madison, WI), water from Milli-Q Ultrapure Water system (Millipore, Billerica, MA), and 18Olabeled water (95% isotope purity) from Euriso-top (Gif sur
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Proteomics Based on Peptide Fractionation by SDS-Free PAGE Table 1. Streptokinase Tryptic Peptide Fractionation za
z/m
372
IITVYMGK SKPFATDSGAMPHKLEK77b 275 YYVLK279
-0.002 -0.004 -0.09
-0.0020 -0.0021 -0.1353
365 275
IITVYMGK372 YYVLK279
-0.002 -0.09
-0.0020 -0.1353
3
38
-0.92
-0.5483
4
38
FFEIDLTSRPAHGGK52 280 KGEKPYDPFDR290 123 DGSVTLPTQPVQEFLLSGHVR143
-0.92 -1.06 -1.87
-0.5483 -0.7838 -0.8203
5
280
KGEKPYDPFDR290 TLAIGDTITSQELLAQAQSILNK210 123 DGSVTLPTQPVQEFLLSGHVR143 211 THPGYTIYER220 336 LLYNNLDAFGIMDYTLTGK354 1 MIAGPEWLLDRPSVNNSQLVVSVAGTVEGTNQDISLK37 (deamidated)
-1.06 -1.91 -1.87 -1.05 -2.09 -3.92
-0.7838 -0.7869 -0.8203 -0.8510 -0.9679 -0.9953
373
RPEGENASYHLAYDKDR389 EVYSYLR402 234 TILPMDQEFTYHVK247 b 373 RPEGENASYHLAYDK387
-2.11 -1.05 -2.00 -2.10
-1.0440 -1.1349 -1.1611 -1.2001
158
SVDVEYTVQFTPLNPDDDFRPGLK181 DGSVTLPTQPVQEFLLSGHVR143 (deamidated) 321 NLDFR325 403 YTGTPIPDNPNDK415 336 LLYNNLDAFGIMDYTLTGK354 (deamidated) 300 YVDVNTNELLK310
-3.01 -2.87 -0.87 -2.00 -3.09 -2.00
-1.0942 -1.2583 -1.3142 -1.3982 -1.4297 -1.5308
300
-2.00 -3.11 -1.87 -3.10 -3.16 -7.01
-1.5308 -1.5381 -1.6523 -1.7706 -1.8169 -1.8392
-2.87 -6.96 -3.00 -3.91 -3.00 -1.96 -3.87 -4.91
-1.8901 -1.9829 -2.0952 -2.2022 -2.2937 -2.5232 -2.5469 -2.7637
gel slice
1
peptide sequence 365 61
2
FFEIDLTSRPAHGGK52
188
6
396
7
123
8
YVDVNTNELLK310 RPEGENASYHLAYDKDR389 (deamidated) 311 SEQLLTASER320 373 RPEGENASYHLAYDK387 (deamidated) 390 YTEEEREVYSYLR402 83 AIQEQLIANVHSNDDYFEVIDFASDATITDRNGK116 (deamidated) 221 DSSIVTHDNDIFR233 83 AIQEQLIANVHSNDDYFEVIDFASDATITDR113 403 YTGTPIPDNPNDK415 (deamidated) 259 SGLNEEINNTDLISEK274 300 YVDVNTNELLK310 (deamidated) 326 DLYDPR331 221 DSSIVTHDNDIFR233 (deamidated) 259 SGLNEEINNTDLISEK274 (deamidated) 373
a z is the calculated valence according to the Sillero and Ribeiro equation.27 methionine.
Yvette, France). Escherichia coli strain W3110 cells were processed by sequential solubilization as described by Molloy et al.,9 and the soluble fraction in Tris was stored at -20 °C. Outer membrane vesicles obtained as a previous step to the active pharmaceutical ingredient of VA-MENGOC-BC (Neisseria meningitidis serogroup B strain CU385) was purchased from Finlay Institute (Cuba). Bio-Rad Protein Assay kit (Hercules, CA) was used for Bradford protein content estimation. 2.2. Protein Fractionation by Polyacrylamide Gel Electrophoresis. Soluble proteins extracted in Tris from E. coli (0.01 or 0.3 mg for analytical and preparative gels, respectively) and N. meningitidis outer membrane preparation (0.03 or 0.5 mg for analytical and preparative gels, respectively) were reduced in 1% dithiothreitol (DTT), 2% sodium dodecyl sulfate (SDS), and 50 mM Tris-HCl, pH 8.8, during 1 h at 37 °C, and cysteines were blocked with 2.5% acrylamide during 1 h at 25 °C. rSK (1 µg) was diluted in nonreducing Laemmli buffer.1 Gel electrophoresis of proteins in the presence of SDS (70 × 70 × 0.75
b
These peptides were also detected as peptides containing oxidized
mm or 70 × 70 × 3 mm) and gel staining with Coomassie Brilliant Blue G-250 (CBB) were done according to standard procedures. For preparative purpose, gels were not stained and the lane was cut into 3 mm bands (16 segments). Bands were washed once in 1 mL of water and twice in 1 mL of 30% acetonitrile (ACN) during 10 min. 2.3. In-Gel Digestion. Bands were cut in 1 mm3 pieces, dehydrated with 90% ACN, rehydrated in a minimum volume of 25 mM ammonium hydrogen carbonate (AmBC) containing modified trypsin (12.5 ng/mL), and digested at 37 °C for 16 h. In some experiments, trypsin and AmBC were dissolved in H218O (95% isotope purity). Digestion in H218O was stopped by reducing trypsin in 2% β-mercaptoethanol and alkylating with 5% acrylamide. 2.4. Peptide Transfer to the SDS-Free PAGE. Two different approaches (“in-solution loading” and “in-gel loading”) were evaluated to determine the transfer efficiency of the tryptic peptides to the SDS-free gel. Journal of Proteome Research • Vol. 7, No. 6, 2008 2429
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Figure 2. Peptide fractionation by PAGE. Expanded region of mass spectra for rSk tryptic peptides separated in a 15% PAGE. The lane was cut in 8 slices. (A) unfractionated peptide mixture; (B-E) peptides obtained from slices 5-8, respectively.
2.4.1. In-Solution Loading. Peptides were eluted in 100 µL of 25 mM AmBC during 45 min, then in 100 µL of 5% formic acid. Both solutions were mixed and concentrated under vacuum up to 10 µL (for direct mass spectrometry analysis samples were concentrated up to 50 µL). The concentrated peptide solution was diluted 2-fold in 125 mM of Tris-HCl buffer, pH 6.8, and 25% glycerol and loaded to the SDS-free gel. 2.4.2. In-Gel Loading. Gel pieces containing the digested proteins were incubated for 15 min at 25 °C in 10-15 µL of 500 mM Tris-HCl, pH 6.8, and 50% glycerol. The gel blocks as well as any surrounding solution were placed into the well of the preparative minigel (0.3 mm thick) and covered with electrode buffer. 2.5. Electrophoretic Peptide Fractionation. The discontinuous buffer system described by Ornstein2 was used. It consists on a 15% acrylamide-bisacrylamide separating gel (3% C), 375 mM Tris-HCl buffer, pH 8.8, and a 6% acrylamide-bisacrylamide stacking gel (3% C), 125 mM Tris-HCl buffer, pH 6.8. The peptide digest was loaded either in solution or in gel as described previously. Electrode buffer solution of 25 mM Tris and 192 mM glycine was used. Bromophenol blue (BPB) was applied in lanes adjacent to the sample and the electrophoretic run was stopped just before BPB reached the gel bottom. The lane was cut into 8 slices (1 × 6 mm or 3 × 6 mm) and peptides were eluted as described in section 2.4.1. Phosphopeptides from casein were isolated with TiO2 Glygen (Columbia, MD) microcolumn, according to manufacturer’s instructions. 2.6. Mass Spectrometry. Peptides were separated by nanoHPLC (Agilent 1100 Series) using an RP-C18 column, 75 µm i.d. × 150 mm (Zorbax) connected online to a hybrid quadrupole orthogonal acceleration tandem mass spectrometer QTof-2 (Micromass, Manchester, U.K.). Capillary and cone voltage of 2000 and 35 V, respectively, were used. The spectra were acquired in the m/z range from 400 to 2000 Th. Peptides were eluted from the column using a lineal gradient of acetonitrile (5-35%) with 0.1% of formic acid in 60 min, at 200 nL/min. 2430
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Ramos et al. The precursor ions were selected within a window of 4 Th. Doubly- and triply charged precursor ions to be fragmented were selected automatically once their intensity rose above 10 counts/s. Data acquisition and processing were performed using a Masslynx system (version 3.5) from Micromass (Manchester, U.K.). Protein identification based on MS/MS spectra was made using Internet-available search engine MASCOT (http://www.matrixscience.com). All the LC-MS/MS data (pkl files) coming from the same SDS-PAGE band are submitted at once. Variable modifications including deamidation of Gln and Asn, and methionine sulfoxide were taken into account. Other restrictions to MASCOT were precursor ion m/z tolerance of 0.2 Da, enzyme digestion with trypsin, up to one missed cleavage, and only doubly- and triply charged precursor ions were considered. The ESI-MS/MS were also manually inspected and considered as reliable identifications when four or more consecutive C-terminal yn′′ fragment ions were assigned to intense signals. Theoretical isoelectric points were obtained with the InSilicoSpectro,20 and GRAVY values were obtained from ExPASy (http://ca.expasy.org/tools/protparam.html). 2.7. Validation. Protein sequence databases were downloaded from the Universal Protein Resource (Uniprot) Web site (http://www.pir.uniprot.org/index.shtml), release 12.5 (13-Nov2007). Peptide/protein identifications were performed with the Mascot search engine (http://www.matrixscience.com/). The Mascot ion-score was used as the metrics to rank the peptides candidates hits. A fair comparison between the method proposed here and the direct analysis of the digested-protein band18 is intended by using the target-decoy strategy.21 The performance is assessed in terms of the false positive (FP) rate and sensitivity estimations of both methods. MS/MS data files were searched against a composite target-decoy protein sequence database.22 The target entries comprise all the E. coli protein sequences found in the Uniprot database. The decoy entries comprise the reversed sequences of all target-proteins entries. The targetdecoy strategy relies on the assumption that true-positive (TP) peptide identifications are only assigned to peptides from target entries in the database, while FP peptide identifications are indistinguishably assigned by chance to a target or a decoy entry. The peptide FP rate identifications were estimated as twice the number of decoy entry hits divided by the total number of peptide identifications in the data set. The estimation of protein identification FP rate was based on the same calculation.
3. Results and Discussion 3.1. Peptide Fractionation by SDS-Free PAGE. The Ornstein gel electrophoresis system,2 although described many years ago for protein separation, has not been used as the separating tool for peptide fractionation in proteomics studies. In the absence of SDS, the driving force for the electrophoretic migration toward the anode is supplied by negatively charged acid amino acid residues and other residues as phosphate, sulfate and sialic acid, while the resulting mobility depends on both the charge and the molecular mass of the peptides. Streptokinase is a 47 kDa protein that contains 29 theoretical tryptic peptides with masses between 600 and 4000 Da; it was used as a model for evaluating the electrophoretic fractionation of peptide digests in the absence of SDS. A tryptic digest from rSK was separated in a 15% PA gel, the unstained gel lane was cut into 8 slices (Figure 1), and the peptides in each slice were recovered by
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Proteomics Based on Peptide Fractionation by SDS-Free PAGE
Figure 3. The most acidic peptides and phosphopeptides are detected in gel slices corresponding to the fastest migration. Tryptic peptides from a mixture of caseins were separated in a 15% acrylamide PAGE. The lane was cut in 8 slices. (A) Unfractionated peptide mixture; (B) peptides identified in the slice 7; (C) peptides identified in the slice 8; (D) peptides isolated using titanium dioxide affinity chromatography, Asterisk (*) phosphopeptides. Mass values 964.38 and 1049.78 corresponded to di- and tetraphosphorylated peptides.
Table 2. Number of Peptides and Proteins Identified in the Slices 7 and 11 slice 7
Without peptide fractionation With peptide fractionation
slice11
no. identified proteins
no. assigned peptides
no. peptides/ no. proteins
no. identified proteins
no. assigned peptides
no. peptides/ no. proteins
22 57
155 282
7.0 4.9
31 85
119 276
3.8 3.2
passive diffusion and were analyzed by nanoESI-MS (Table 1). A total of 33 peptides (including peptides containing one missed cleavage) distributed in 8 slices allowed the automatic identification of rSK with an 83% of sequence coverage using the “peptide mass fingerprint” tool. Twenty-seven peptides were found in unique gel slices (for 82% fractionation selectivity with respect to the total assigned peptides). Six peptides were found in two consecutive slices. Figure 2 shows an expanded region of the mass spectrum of the resultant (unfractionated) digestion and those corresponding to consecutive gel slices 5, 6, 7, and 8 (see Supporting Information for all slices). The mass spectrum of the total digestion (Figure 2A) allows the detection of only one signal (m/z 761.08) with a complex isotopic ion distribution that was resolved into three signals after peptide fractionation (Figure 2B-E). The triply charged signals at m/z 760.73 and 761.06 corresponding, respectively, to peptide 123DGSVTLPTQPVQEFLLSGHVR143 and its deamidated form and the doubly charged signal 759.86 corresponding to peptide 221DSSIVTHDNDIFR233 were detected in the slices 5, 7, and 8, respectively. The intensity of the deamidated peptide with respect to the normal one 123 D-143R (m/z 760.73) is 100-fold lower; and the mass spectrometer was able to detect and sequence it only after fractionation. Deamidation of glutamine is less favored, which is consistent with the deamidated ratio found. For several peptides, the procedure separated in different slices the deamidated peptides and their normal (amidated) forms (data not shown), the firsts with faster migration as consequence of the
increase in the acidic content with an increment in the their effective negative charge; therefore, the technique could be particularly useful for studying peptide deamidation. The signal 754.38 corresponding to peptide 236LPMDQEFTYHVK247 was hardly observed in the original sample (Figure 2A); however, it appears as an intense isotopic cluster after fractionation (Figure 2C). This result points out the utility of this procedure for the detection and identification of low-abundance proteins. In this electrophoretic system, peptides having a negative net charge at alkaline pH migrate to the anode, with a migration rate directly proportional to their effective charge and inversely proportional to their frictional resistance to the medium.23 For this reason, peptides with high content of acidic amino acids and substituents are expected in gel slices corresponding to the fastest migration. Phosphopeptides frequently have an acidic isoelectric point due to the phosphate group linked to serine, threonine or tyrosine residue. To analyze the behavior of this kind of peptides, we applied this procedure for fractionating a tryptic digest from a mixture of alpha S1, alpha S2 and beta caseins. In this case, gel slice number eight (corresponding to the fastest migration) was cut exactly with the dimensions of the adjacent bromophenol blue reference. Interestingly, in this gel slice, all the phosphopeptides detected by using the titanium oxide approach24 were also detected after PAGE fractionation (Figure 3). Five phosphopeptides were identified, three of them are monophosphorylated, a fourth peptide was found mono- and diphosphorylated, and the fifth one was identified carrying four phosphate groups; this peptide Journal of Proteome Research • Vol. 7, No. 6, 2008 2431
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Figure 4. Proteins are separated according to their molecular mass and peptides are separated according to their charge/mass ratio. 9, Histogram of mass value for proteins identified in the band 11; 2, histogram of mass value for proteins identified in the band 7; (, theoretical valence/mass ratio (z/m) for peptides identified in each slice in the SDS-free gel. Peptides from slices 1 and 8 were not included; the estimated valence/mass ratio for these peptides and their mobility did not show a linear correlation. z is the calculated valence according to the Sillero and Ribeiro equation.27
Figure 5. Identification of low-abundance proteins from a membrane protein extract. The approach was applied to the outer membrane vesicles prepared from N. meningitidis (lane I). Subcellular location were obtained from PSORTb (www.psort. org/psortb/). a, Iron-regulated outer membrane protein; b, outer membrane protein class 1; c, outer membrane protein class 3 and 4.
was not identified after selective isolation using titanium oxide minichromatography. Several signals corresponding to nonphosphorylated peptides were also detected in both approaches. 2432
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Ramos et al. There is a previous report from Gatti and Traugh25 for a method of phosphopeptide separation using a two-dimensional peptide electrophoresis system that combines nondenaturing isoelectric focusing and alkaline electrophoresis using a 40% polyacrylamide gel. Because of the high density of the seconddimension gel, there is a significant sieving effect provoking phosphopeptides to be found through the gel. In the present approach, we use an alkaline 15% polyacrylamide gel with negligible sieving effect for peptides. In consequence, peptides with high content of acidic residues comigrate with phosphopeptides in the fastest moving fraction. This class of peptides also appears as contaminants after selective phosphopeptide enrichment methods such as titanium oxide and immobilized metal ion affinity chromatography (IMAC).26 Therefore, further studies must be done to demonstrate the applicability of this procedure to phosphoproteomics. 3.2. Applications in Proteomics. In 2001, Simpson et al. proposed the combination of SDS-PAGE and LC-MS/MS strategy for proteomics studies and identified 284 proteins from a crude plasma membrane of the human colon carcinoma cell line LIM1215.18 However, peptides with low relative abundance that coeluted with other peptides present at higher levels were not amenable to the identification; thus, the authors suggested an additional peptide fractionation to overcome this limitation. Now, peptide fractionation in SDS-free PAGE was combined with a previous separation of proteins by SDS-PAGE, a technique particularly convenient for the fractionation of hydrophobic proteins. Soluble proteins (300 µg) obtained after E. coli cell disruption were fractionated by SDS-PAGE and the lane was cut in 16 segments. Tryptic digests from gel segments 7 and 11 (arbitrarily selected) were fractionated by SDS-free PAGE after ingel loading, the corresponding lanes were cut into 8 slices each, and peptides were eluted and separately analyzed by nanoLCMS/MS (Figure 1). Compilation of the eight LC running for a single database query enhances the MASCOT score and, therefore, the reliability of protein identification. The assigned peptides were 282 and 276 for segments 7 and 11, which permitted the identification of 57 and 85 proteins, respectively. Gel segments 7 and 11 from an adjacent lane were also digested with trypsin and analyzed directly by nanoLC-MS/ MS, as previously described by Simpson,18 to compare with these results. The number of assigned peptides and identified proteins were 2.3- and 2.7-fold higher after peptide fractionation of fraction 11 (Table 2) and 1.8- and 2.6-fold higher for fraction 7. All the proteins identified by direct analysis of the peptide mixture were also identified when peptide fractionation via SDS-free PAGE was performed. In the SDS gel bands 7 and 11, 35 and 55 new proteins were additionally identified after peptide fractionation, respectively. These results were obtained after manual inspection of MASCOT results. To validate this analysis, a statistical assessment of peptide assignment and protein identification by using target-decoy was applied. Considering a FP rate of 2%, twice more peptides and proteins were identified after peptide fractionation (similar to manual inspection). All the peptides identified after fractionation have a calculated pI lower than 7.35. As only negatively charged peptides migrate to the anode in this electrophoresis system, there is a simplification of peptide digests (in this example, 4 peptides/ protein in comparison to 5.2 peptides/protein after the direct analysis18 were found as average); this surrogate of the peptide digests allows a more efficient MS/MS analysis while keeping
research articles
Proteomics Based on Peptide Fractionation by SDS-Free PAGE a confident identification. In silico analysis revealed that 97% of the E. coli proteome can be analyzed by selecting peptides with pI lower than 7.35, which simplifies the complex peptide mixture to 9 out of 11 theoretical tryptic peptides per protein. However, the set of theoretical peptides amenable to analysis can be modulated by the pH of the discontinuous buffer system used for the SDS-free PAGE peptide fractionation. In fact, West et al.19 described the usefulness of acidic gel with an operative pH between 3.0 and 3.1 for the analysis of positively charged peptides. An electrophoretic system with a central sample application point permitting peptides migration to both, anode and cathode, through the gel could also be devised. A majority of the identified proteins in each SDS-gel band have a molecular mass in the expected mass range according to their electrophoretic migration in the SDS gel. For the SDS gel band 11, 92% of the 85 identified proteins have mass values between 20 and 32 kDa (Figure 4, top panel), while only 7 were out of this expected mass range (5 were higher and 2 were lower than the apparent mass according to electrophoresis). Protein degradation and anomalous electrophoretic mobility described for SDS-PAGE could explain these results. Similar results were observed for proteins present in band 7. In SDS-free PAGE, migration of peptides is a function of their net charge and the molecular size. For convenience, charge of peptides is generally expressed in terms of calculated valence z, that can be determined from the amino acid sequence,23,27 while molecular mass is used instead of molecular size. Migration or electrophoretic mobility is then directly related to the estimated valence and inversely related to the molecular mass. Considering the set of peptides identified in the successive slices, although we did not determine the Rf for each individual peptide inside the slice, we found a correlation value of 0.9051 between the estimated charge-to-mass ratio for the peptides and their mobility (Figure 4, bottom panel). These results suggest that, in addition to the protein mass estimated from the SDS-PAGE, the calculated pI and the theoretical electrophoretic mobility can also contribute to validate mass spectrometry identifications. Peptide transfer from the first step (SDS-PAGE) to the second step (SDS-free PAGE) is a critical point to obtain the highest recoveries. Two strategies were evaluated for loading tryptic digests into the SDS-free gel: (a) peptides were passively eluted by diffusion from the gel slice, the eluate was concentrated and loaded into a 0.75 mm thick gel (in-solution loading), (b) the small gel pieces containing digested proteins were loaded into the well of a 3 mm thick gel (in-gel loading) and tryptic peptides were electrophoretically transferred during the separation. To compare the efficiency of both procedures, rSK was digested in-gel in normal water and the peptides eluted by diffusion were in-solution loaded into a 0.75 mm thick gel. Alternatively, rSK was digested in-gel in 18O-labeled water and the gel blocks containing the digested protein were loaded in a 3 mm thick gel. After electrophoresis, the lanes were cut into 8 slices, peptides were independently eluted, and both eluates were mixed and analyzed by nanoLC-ESI-MS. Even when lower recovery of peptides could be expected from a thicker gel, the general recovery after in-gel loading and electrophoretic transfer was higher than after in-solution loading of passively eluted peptides. All the isotopic distributions show higher intensity (more than 2-fold) for the 18Olabeled peptides, reflecting losses during peptide elution and concentration for in-solution loading. In fact, losses of peptide digests up to 40% have been reported during passive elution
followed by vacuum concentration of the extracts.28 In consequence, to achieve a high peptide recovery, peptide electrotransfer to the SDS-free gel is preferred. 3.3. Identification of Low-Abundance Components in a Membrane Protein Extract. Some biological samples are considered as particularly challenging for proteomic technologies, a paradigmatic case being animal sera, where a few components represent about 90% of the protein mass. Another highly challenging sample is the active ingredient of the vaccine VA-MENGOC-BC against N. meningitidis: the sample is rich in membrane proteins, there is a high content of lipids and four proteins (outer membrane protein class 1, class 3, class 4 and the Iron-regulated outer membrane protein) constitute about 70% of the protein mass. Additionally, the degradation products derived from these four proteins are at the same level or higher than the low represented proteins.29 Because of these properties, its study represents a major challenge for 2DE and for other methodologies in proteomics. This complex sample has been the subject of previous research in our group using bidimensional electrophoresis/mass spectrometry approach29 and also applying the new methodology SCAPE30 for the selective capture of peptides (unpublished results). Here, we applied the present method to the analysis of the active component of the N. meningitidis preparation (Figure 5). In a Coomassie blue stained SDS gel, a few bands are observed, all corresponding to the dominant components of the preparation. A total of 97 proteins were identified, 31 membrane proteins and 37 citosolic proteins (Supporting Information). Interestingly, we identified several membrane proteins that have been evaluated by other authors as candidate for N. meningitidis type B vaccine. This is the case for NMB039431 and NMB046132 among others. In this approach, the presence of SDS in the initial extraction solution allows the solubilization of most proteins including those highly hydrophobic. Here, we identified 10 proteins with a positive GRAVY index, with the phosphoglycerate kinase as the most hydrophobic protein (GRAVY: 0.180).
4. Conclusions The high complexity of the proteome justifies the combination of several successive separation procedures for increasing the number and the quality of protein identifications following the conventional bottom-up approaches.33 For being mutually potentiated, these separation procedures should be based on orthogonal principles. Even after multiple fractionations, the number of proteins/peptides in a fraction remains so important as to evade full identification by conventional procedures based on LC-MS/MS. An additional advantage can be obtained if one of the procedures selects a surrogate of peptide digests. The method here presented, based on SDSfree PAGE of protein digests, allows the selection of acidic peptides as a surrogate for the entire digest. These peptides migrate to the anode, the high majority being identified in only one SDS-free gel slice. As frequent post-translational modifications introduce acidic groups (phosphate, sulfate, sialic acid), the procedure can be applied to the detection of the corresponding modified peptides. By combining this procedure with a previous electrophoretic fractionation of proteins in SDS gel, a higher number of proteins can be identified. The method uses common electrophoretic equipments available in any protein chemistry laboratory. Quantitative comparison of proteomes for two or more conditions can easily be adapted by using Journal of Proteome Research • Vol. 7, No. 6, 2008 2433
research articles differential protein labeling procedures (SILAC, TMT, etc.) before protein digestion.
Acknowledgment. This work was granted by the research program of the Centre for Genetic Engineering and Biotechnology (Havana, Cuba). We thank Darien García and Daniel Yero for the fruitful scientific discussions. Supporting Information Available: Mass spectra of streptokinase trypsin digest after peptide fractionation, the proteins identified on the N. meningitidis preparation. This material is available free of charge via the Internet at http:// pubs.acs.org. References (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14)
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