From the Institut de Pharmacologie et de Biologie Structurale, Centre National de la Recherche Scientifique, 205 route de Narbonne, 31077 Toulouse cedex, France, and ¶ Centre de Recherche en Pharmacologie-Santé, CNRS-Pierre Fabre, 205 route de Narbonne, 31077 Toulouse cedex, France
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ABSTRACT |
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Protein isoforms may originate from alternative splicing of mRNA, single-point mutations, and post-translational modifications including proteolytic cleavages. These modifications often introduce a variation in the molecular mass and net charge of the protein. The most efficient technique to separate protein isoforms thus remains 2D gel electrophoresis. The identification and localization of the modification can then be obtained using MS/MS analyses of the peptide bearing the modification after proteolysis of the protein. Depending on the nature of the modification to be identified, different strategies to detect the modified peptide can be employed. The choice of this strategy would be facilitated if the molecular mass of the intact protein isoform were known. Indeed, the mass difference between two isoforms or between the isoform and the expected theoretical molecular mass would allow drawing a solid hypothesis on the nature of the protein modification. Efforts have been made to extract proteins from 2D polyacrylamide gels using various methods, including electroelution (1215), electrotransfer on membranes (1618), and passive elution (1921). These methods, however, are not adapted for the further determination of the molecular mass of picomoles of protein isoforms using MS. For example, electroelution uses large elution volumes leading to significant losses of material. Electrotransfer on nitrocellulose and polyvinylidene difluoride membranes is compatible with matrix-assisted laser desorption/ionization time-of-flight analysis of intact proteins, but mass accuracy is not always sufficient using this technique for the identification of protein modifications. Finally, passive elution has been described using formic acid, which may lead to formylation of serine and threonine residues (20), thus excluding the use of this approach for post-translational modifications studies. Passive elution has also been described using SDS (22). The presence of this detergent being detrimental for MS analyses, a further step to remove SDS is necessary to allow MS analyses.
Here we report on the usefulness of a strategy combining the "bottom up" and "top down" approaches to characterize protein isoforms resulting from single-point mutations and post-translational modifications. This strategy is based on polyacrylamide gel electrophoresis separation of protein isoforms, MS and MSn analyses of intact proteins, and MS/MS analyses of proteolytic peptides. Protein isoforms are extracted from polyacrylamide gels by passive elution using SDS, followed by nanoscale hydrophilic phase chromatography for SDS removal. Electrospray ionization (ESI)-MS analyses of the intact proteins are performed to determine their molecular mass, allowing hypotheses on the nature of the modification to be drawn. In the case of labile post-translational modifications, like phosphorylations and glycosylations, ESI-MSn analyses of the intact proteins are conducted to confirm their presence. Finally, proteins are digested in solution, and MS/MS analyses of the modified peptides are performed to locate the modifications. This strategy allows the determination of the molecular mass of 510 pmol of a protein up to circa 50 kDa loaded on a gel. The efficiency of this approach for the characterization of protein variants and post-translational modifications is illustrated with the study of -casein isoforms.
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MATERIALS AND METHODS |
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2D Gel Electrophoresis
-Casein (Sigma Aldrich) was desalted by trichloracetic acid precipitation and resuspended in the rehydration buffer (9 M urea, 2.2% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid, 65 mM dithiothreitol). The first dimension was performed on the IPGphor system (Amersham Pharmacia Biotech). Briefly, the 7-cm pH 47 immobilized pH gradient strip (Amersham Pharmacia Biotech) was rehydrated with the sample solution for 11 h under 50 V at 20 °C. Then the sample was focused for 30 min at 200 V, 30 min at 500 V, 1h at 2000 V, followed by 8000 V for a total of 80 kVh.
Immediately prior to the second dimension, the IPG strip was incubated in 5 ml of 50 mM Tris-HCl, 6 M urea, 2% SDS, 30% glycerol, and 65 mM dithiothreitol under rotary shaking for 15 min, followed by 15 min in the same solution where dithiothreitol was replaced by 2.5% iodoacetamide.
SDS-PAGE was performed on the MultiPhor II system using a 12.5% acrylamide precast gel (Amersham Pharmacia Biotech). Complete migration was achieved at 15 °C after 1 h at 200 V followed by 600 V for a total of 500 Vh. Spots were visualized by Coomassie Brilliant Blue staining.
Passive Elution of Proteins from Polyacrylamide Gels
Gel pieces were excised and washed with H2O for 2 h and proteins were allowed to diffuse out of the gel overnight at 37 °C by incubation in 30 µl of 0.1 M sodium acetate, 0.1% SDS, pH 8.2.
SDS and Salts Removal Using Hydrophilic Phase Chromatography
Coomassie Brilliant Blue, SDS, and salts were removed from the protein sample after passive elution using a ZipTipHPL (Hydrophilic Interaction Chromatography) according to the manufacturers instructions (Millipore, Bedford, MA). Briefly, the ZipTipHPL is rehydrated in buffer B (H2O/ACN/AcOH 50/50/0.1, pH 5) and washed with buffer A (H2O/ACN/AcOH 10/90/0.1, pH 5.5). Protein eluates are diluted in 200 µl of buffer A and are loaded onto the ZipTipHPL. Salts are removed by washing with buffer A, and proteins are eluted in 4 µl of H2O/ACN/TFA 50/50/0.1.
Nano-ESI-MS of Intact Proteins
Nano-ESI-MS analyses were performed on an ion trap mass spectrometer (LCQ Deca, ThermoFinnigan, San Jose, CA). Mass spectra were acquired by scanning an m/z range from 370 to 2000 at 70 ms/scan. Eluates (0.5 µl) were injected into the mass spectrometer using the Famos® auto-sampler (LC Packings, Dionex, Amsterdam, The Netherlands) and the Ultimate® pumping device (LC Packings) through a fused silica capillary (ID 75 µm) at a flow rate of 250 nl/min (carrier solvent: H2O/MeOH/acetic acid 50/50/0.5). Electrospray needles were obtained from New Objective (Woburn, MA). Spray voltage was set at 1.8 kV, and capillary temperature was set to 180 °C. Mass spectra were deconvoluted using the Biomass® program included in the Xcalibur® software (ThermoFinnigan).
For MSn experiments, various mass-to-charge ratios were selected using a 1 m/z unit ion isolation window and fragmented by collisions using a 20% relative collision energy and a Q activation parameter of 0.25 for 30 ms.
On-line Capillary High-pressure Liquid Chromatography-Nanospray MS/MS Analyses of Protein Digests
After extraction of the protein from the gel spot and desalting, acetonitrile was evaporated from the eluate by vacuum centrifugation. Five microliters of trypsin (Promega, Madison, WI) at 12.5 ng/µl were added, and the sample was incubated at 37 °C for 4 h. The peptide mixture was directly analyzed by on-line capillary high-pressure liquid chromatography (LC Packings) coupled to a nanospray ion trap mass spectrometer (LCQ Deca, ThermoFinnigan). Peptides were separated on a 75 µm ID x 15 cm C18 PepMapTM column (LC Packings). The flow rate was set at 150 nl/min. Peptides were eluted using a 0 to 40% linear gradient of solvent B in 40 min (solvent A was 0.1% formic acid in 2% acetonitrile, and solvent B was 0.1% formic acid in 90% acetonitrile). The mass spectrometer was operated in positive ion mode at a 1.8-kV needle voltage and a 30-V capillary voltage. Data acquisition was performed in a data-dependent mode consisting of, alternatively in a single run, a full scan MS over the range m/z 3702000, a zoom scan of the ion selected in an exclusion dynamic mode (the most intense ion is selected and excluded for further selection for a duration of 0.5 min), and a full scan MS/MS of the selected ion. MS/MS data were acquired using a 3 m/z units ion isolation window and a 35% relative collision energy.
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RESULTS |
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Myoglobin (17 kDa), carbonic anhydrase (29 kDa), protein A (45 kDa), and bovine serum albumin (64 kDa) were used as standard proteins. One microgram of each protein was loaded onto a polyacrylamide gel and submitted to electrophoresis. After Coomassie Blue staining, spots were excised from the gel and submitted either to passive diffusion alone or to passive diffusion followed by SDS removal. Each sample was then submitted to a subsequent migration by SDS-PAGE, and quantities of proteins were calculated based on the corresponding band intensity after Coomassie Blue staining. Protein elution from the gel was performed in the presence of 0.1% SDS. Further SDS removal from the protein samples is a critical step to allow ESI-MS analysis. It has been achieved using ZipTipHPL chromatography, a subset of normal phase chromatography. Separation depends on hydrophilic interactions between the solutes and the hydrophilic stationary phase. Originally designed for detergent and Coomassie Blue stain removal (not retained) from peptide mixtures, the ZipTipsHPL are well suited for amounts of sample in the microgram range. The protocol was optimized for protein samples using trifluoroacetic acid instead of formic acid in the elution buffer.
As shown in Fig. 1, proteins up to 45 kDa efficiently diffused out of the gel and were recovered from the ZipTipHPL. In contrast, bovine serum albumin (64 kDa) could not be extracted from the polyacrylamide gel. Based on spot intensities after Coomassie Blue staining, the yield of protein recovery for myoglobin, carbonic anhydrase, and protein A reached 60% after passive elution and 30% after the additional SDS removal step.
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Forty micrograms of a -casein isoform mixture were separated by 1D and 2D SDS-PAGE. As shown in Fig. 3, the intense band detected around 22 kDa after 1D SDS-PAGE splits into several spots (numbered from 1 to 6) with similar apparent molecular mass but with isoelectric point varying from 5 to 6.3 after 2D SDS-PAGE. These spots were all identified as
-casein by the "bottom-up" proteomic approach. Two additional spots of lower molecular mass were also observed and identified as ß-lactoglobulin (spots 7 and 8).
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It is noteworthy that the measured mass of an intact protein may not necessarily correlate with the predicted mass obtained from the sequence data base, which contains possible errors in protein sequences and does not integrate post-translational modifications. If observed, the mass difference between two protein isoforms may then allow the identification of a modification without having the complete sequence information.
Post-translational Modification Characterization Using ESI-MSn Analyses of Intact -Casein Isoforms
The determination of the molecular mass may not always be sufficient to identify protein modifications, in particular when there is a combination of modifications and when the mass difference with the theoretical molecular mass is important as it is the case with the -casein isoform from spot 6. Moreover, the micro-amounts of protein present in the spot eluates did not allow the use of biochemical studies to confirm the presence of phosphorylations and to elucidate the saccharidic motif suspected in spot 6. In order to evidence the presence of such post-translational modifications, we took advantage of the capabilities of an ion trap mass spectrometer and investigated the efficiency of ESI-MSn analyses of gel-eluted proteins.
MS/MS experiments were initially conducted on -casein eluates from the previously analyzed spots 1 to 6. Fig. 5 presents the MS/MS spectrum of the 10-charge precursor ion from spot 2 (Fig. 4B, m/z 1912.810+), from which molecular mass measurement indicated the presence of a phosphorylation. This spectrum is dominated by peaks close to the precursor ion at m/z 1910.9, 1909.2, and 1902.9. Assuming that these ions possess 10 charges, the major peak at m/z 1902.9 corresponds to the loss of one phosphate group from the precursor protein (98 Da, i.e. H3PO4). The other peaks (m/z 1910.9 and m/z 1909.2) correspond to successive losses of water molecules from the precursor ion. MS/MS experiments conducted on other charge states of the protein led to identical results (data not shown). These MS/MS analyses thus confirm that the
-casein isoform from spot 2 is monophosphorylated and show the ability of ESI-MS/MS experiments of gel-eluted proteins to highlight the presence of a phosphorylation.
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The approach was then applied to the -casein isoform from spot 6, for which molecular mass measurement did not readily identify its modifications. The ESI-MS2 spectrum of the 12-charge precursor ion (m/z 1675.812+) of the 20099-Da unidentified isoform from spot 6 is presented in Fig. 6A. The spectrum displays several major peaks at m/z 1674.2, 1651.1, 1650.1, 1626.9, and 1626.0. Considering that all of these ions possess 12 charges, they correspond to increasing losses of 19, 296, 308, 587, and 598 Da, respectively, from the precursor protein. These losses can be attributed to neutral losses of one H2O molecule, one N-acetyl neuraminic acid moiety (NeuAc), one NeuAc and one H2O, two NeuAc, and two NeuAc and one H2O, respectively. These data thus show the presence of two NeuAc moieties in the saccharidic motif of the
-casein isoform present in spot 6 (Fig. 3). These results are in good agreement with the NeuAc
(23)Galß (13)[NeuAc
(26)]GalNAc tetrasaccharidic moiety previously described as the major glycosylated moiety in
-casein (27). The protein mass increment due to such a tetrasaccharide is 947 Da, thus bringing the molecular mass of the non-glycosylated form down to 19152 Da. This molecular mass corresponds to the monophosphorylated variant A. To verify the presence of a phosphorylation in addition to the tetrasaccharidic moiety, MS3 experiments were conducted. The fragment ion having lost the two NeuAc groups and one H2O molecule (m/z 1626.012+ in the MS2 spectrum from Fig. 6A) was further fragmented (Fig. 6B). Several fragment ions were observed. Assuming that all fragment ions have the same charge state as the precursor ion, the fragment ion at m/z 1617.3 corresponds to the loss of one phosphate group from the precursor ion. Other fragment ions correspond to losses of one hexose and of one hexose plus an N-acetyl hexosamine. Experiments conducted on other charge states led to identical assignments. These results thus confirm the proposed saccharidic moiety and the presence of an additional phosphorylation of
-casein variant A.
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DISCUSSION |
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A key step in the described approach is the passive diffusion of proteins out of the polyacrylamide gel. The aim was to develop a protein elution procedure applicable after Coomassie blue staining, which remains the most widely used detection system for proteins separated on SDS-PAGE gels. Optimization of the overall diffusion conditions included the removal of SDS prior to MS analyses. This was achieved using hydrophilic phase ZipTipHPL chromatography. Using this procedure, 510 pmol of proteins up to 50 kDa were successfully recovered from the polyacrylamide gel and their molecular mass determined by mass spectrometry. However, higher amounts of material would be necessary for further characterization of the putative modifications. Proteins up to 100 kDa were previously reported to diffuse out of polyacrylamide gels with recovery yields reaching 80% (28). The gels in this case were reverse-stained with the imidazole-zinc system and the elution procedure included more steps. Recently, this approach was applied to the identification of truncated forms of -crystallins (29). The sensitivity of the method presented here and its upper limit in molecular mass may be improved by using less reticulated polyacrylamide gels (<12.5%) and an ultrasonic assisted diffusion step (19, 30), while remaining fast and simple. The sensitivity may also be improved by adding a chromatographic step using a reverse phase C4 column to concentrate the protein sample prior to MS analysis. Recently, an analog of SDS, the acid-labile surfactant, has been shown to facilitate protein identification following polyacrylamide gel electrophoresis compared with the widely used SDS by enhancing peptide recovery (31). The use of this novel surfactant may also improve the recovery of protein elution from the polyacrylamide gel.
A mass accuracy of 0.01% was obtained in all cases for the molecular mass determination of intact proteins by ESI-MS using an ion trap mass spectrometer. This mass accuracy was sufficient to distinguish mutation-based variants of -casein and to detect the presence of post-translational modifications such as glycosylation and phosphorylation. All 2D gel spots of
-casein analyzed were thus identified based on mass spectrometric analyses of intact proteins and their molecular mass measurement. In the case of ß-lactoglobulin, however, the variants differed by only 1 Da and could not be distinguished. A higher resolution analyzer would be required in this case.
The attribution of labile post-translational modifications to -casein variants A and B was ascertained by MS/MS analysis of the intact proteins. The MS/MS parameters were chosen to favor neutral losses corresponding to phosphate groups and glycosydic moieties without fragmenting the protein backbone. Using these conditions the loss of 98 Da, corresponding to the loss of one phosphate group (H3PO4) from a serine residue, was observed for
-casein isoforms in spots 2, 3, and 4. These analyses thus confirm that these isoforms are monophosphorylated and validate the molecular mass-based identification. MS/MS analysis of the isoform from spot 5 also showed the loss of one phosphate group, while the measured molecular mass suggested a diphosphorylated isoform. Due to the low collision energy used in the MS/MS experiment to avoid backbone fragmentation, a further MS step was necessary to observe the loss of the second phosphate group from this protein. These MS3 analyses, readily performed on the ion trap mass spectrometer, could also be performed on the new generations of hybrid instruments such as a Q-trap mass spectrometer.
Linkages engaged by glycosidic moieties and especially by sialic acids are more labile than those engaged by phosphates. This explains the preferential loss of two neutral sialic acids in the MS2 spectrum of the 20099 Da protein in spot 6. The presence of NeuAc has already been evidenced in -casein (32). A disialylated species was previously detected as the major subcomponent of
-casein (27). Moreover, the NeuAc
(23)Galß (13)[NeuAc
(26)]GalNAc tetrasaccharide containing two NeuAc was shown to be the most dominant sugar chain. In order to confirm the suggested glycosidic moiety as well as the identity of the
-casein variant in spot 6, a MS3 spectrum was acquired by fragmenting the de-disialylated fragment in the MS2 spectrum. The observed losses of a phosphoric acid, a hexose, and an acetyl-hexosamine both confirmed the structure proposed for the sugar chain and allowed the identification of the protein as the glycosylated form of the monophosphorylated variant A, in perfect agreement with the molecular mass measured on the intact protein.
In agreement with previously reported data, we confirmed that variants A and B were the major isoforms of -casein and were present in the mixture under various phosphorylation and glycosylation states (24). These results highlight the possibilities offered by the coupling of 2D-PAGE separation with MS and MSn analyses of intact proteins. Indeed, because of the often high heterogeneity of glycosylation, separation of the various isoforms is a key step for the success of their characterization.
To go further in the identification of protein variants and to localize the modifications, MS/MS analysis of the tryptic digest of the protein was performed. After diffusion of the protein out of the polyacrylamide gel and MS analysis of the intact protein, the tryptic digestion was performed in solution. This may represent an advantage over an in-gel digestion for an efficient recovery of all types of tryptic peptides. This strategy was illustrated with the -casein isoform from spot 2, which sequence coverage reached 98%. This sequence coverage included the 53-residue peptide bearing the
-casein phosphorylation. For all monophosphorylated isoforms of
-casein, the MS/MS spectrum of this peptide allowed the localization of the phosphorylation at the same residue, Ser170. These results are consistent with previous studies, which showed that
-casein was fully phosphorylated at Ser170 and only partially at Ser148 (33). Surprisingly, monophosphorylated variant A isoforms appeared at different isoelectric point values on the 2D gel, which could not be explained by differences in the measured molecular masses and by the phosphorylation localization. One hypothesis is that the
-casein mixture contains deamidated isoforms. This modification would explain the isoelectric point shift toward the acidic pH. Moreover, as shown with ß-lactoglobulin, the 1-Da mass increment due to this modification would not be assessed by ESI-MS with a 0.01% mass accuracy.
The strategy reported here aims at characterizing protein variants and post-translational modifications of proteins separated on SDS-PAGE gels by determining the molecular mass of the intact protein. These analyses then took advantage of the MSn capabilities of ion trap mass spectrometers to identify labile modifications, like phosphorylations and glycosylations, directly on the intact protein. This strategy finally allows the digestion of the protein in solution and the analysis of the resulting peptides for the localization of the modification. The determination of the molecular mass of the intact protein is a valuable piece of information for choosing the appropriate strategy for the localization of the modification. The coupling of 2D gel electrophoresis separation to mass spectrometric analyses of gel-eluted intact proteins as described should represent a promising strategy in proteomics.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Published, MCP Papers in Press, June 27, 2003, DOI 10.1074/mcp.T300003-MCP200
1 The abbreviations used are: MS, mass spectrometry; 1D, one-dimensional; 2D, two dimensional; MS/MS, tandem mass spectrometry; ESI, electrospray ionization; NeuAc, N-acetyl neuramininc acid.
* This work was supported in part by grants from the Centre National de la Recherche Scientifique (ACI program Protéomique et Génie des Protéines), the Association pour la Recherche contre le Cancer (Réseau ARECA, Protéomique et Cancer), the Conseil Régional Midi-Pyrénées, the Génopole Toulouse Midi-Pyrénées, and the Consortium Pierre Fabre Médicament (Program ASG, Protéomique et nouvelles cibles thérapeutiques). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Current address: Plate-forme Génomique Fonctionnelle, Université Victor Segalen Bordeaux 2, 146, rue Leo Saignat 33 076 Bordeaux cedex, France.
|| To whom correspondence should be addressed: Institut de Pharmacologie et de Biologie Structurale, Centre National de la Recherche Scientifique, 205 route de Narbonne, 31077 Toulouse cedex, France. Tel.: 33-5-61-17-55-45; Fax: 33-5-61-17-55-49; E-mail: Bernard.Monsarrat{at}ipbs.fr
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REFERENCES |
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