Protein and Proteome Phosphorylation Stoichiometry Analysis by

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Anal. Chem. 2006, 78, 1987-1994

Protein and Proteome Phosphorylation Stoichiometry Analysis by Element Mass Spectrometry Ralf Kru 1 ger,† Dieter Ku 1 bler,‡ Roser Pallisse´,‡ Andreas Burkovski,§,⊥ and Wolf D. Lehmann*,†

Central Spectroscopy and Mechanisms of Biomolecular Interactions, German Cancer Research Center (DKFZ) Heidelberg, Germany and Institute of Biochemistry, University Ko¨ln, Germany

Protein phosphorylation stoichiometry was assessed by two analytical strategies. Both are based on element mass spectrometry (ICPMS, inductively coupled plasma mass spectrometry) and simultaneous monitoring of 31P and 34S. One strategy employs a combination of 1D gel electrophoresis, in-gel digestion, and final µLC-ICPMS analysis (µLC ) capillary liquid chromatography). The other strategy uses the combination of 1D gel electrophoresis, protein blotting, and imLA-ICPMS (imLA ) imaging laser ablation). The two methods were evaluated with standard phosphoproteins and were applied to the analysis of the cytoplasmatic proteome of bacterial cells (Corynebacterium glutamicum) and eukaryotic cells (Mus musculus). The eukaryotic proteome was found to exhibit a significantly higher phosphorylation degree (∼0.8 mol of P/mol of protein) compared to the bacterial proteome (∼0.01 mol of P/mol of protein). Both analytical strategies revealed consistent quantitative results, with the µLC-ICPMS approach providing the higher sensitivity. In summary, two ICPMS-based methods for quantitative estimation of the phosphorylation degree of a cellular proteome are presented which access the native proteome state and do not require any type of label introduction or derivatization. The discovery of reversible protein phosphorylation has unravelled a principle of prime importance in cellular regulation1 and has led to the recognition of the widespread occurrence of kinase/phosphatase-mediated signaling cascades.2-4 Protein phosphorylation analysis was first performed by techniques such as 32P-radiolabeling, antibody technologies, Edman sequencing, or a combination thereof. With the advent of mass spectrometry* Corresponding author. Address: Central Spectroscopy, German Cancer Research Center, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany. Phone: ++49 6221 424563. Fax: ++49 6221 424554. E-mail: wolf.lehmann@ dkfz.de. † Central Spectroscopy, German Cancer Research Center. ‡ Mechanisms of Biomolecular Interactions, German Cancer Research Center. § University Ko ¨ln. ⊥ Current address: Institute of Biology, University Erlangen-Nu ¨ rnberg, Germany. (1) Cohen, P. Nat. Cell Biol. 2002, 4, E127-E130. (2) Blume-Jensen, P.; Hunter, T. Nature 2001, 411, 355-365. (3) Manning, G.; Whyte, D. B.; Martinez, R.; Hunter, T.; Sudarsanam, S. Science 2002, 298, 1912-1934. (4) Johnson, S. A.; Hunter, T. Nat. Methods 2005, 2, 17-25. 10.1021/ac051896z CCC: $33.50 Published on Web 02/14/2006

© 2006 American Chemical Society

based analytical proteomics, tandem mass spectrometry has become a key technology for spotting of phosphorylation sites in proteins,5-8 since it provides reliable structural information at high sensitivity. Although the correlation between MS/MS data and peptide structure is tight and conclusive, the extraction of quantitative information from ESI or MALDI mass spectrometry data requires particular strategic efforts, mostly during the preanalytical steps. Most of these quantitative approaches utilize a stable isotope labeling strategy, which compensates for the sequence-dependent responses of the ESI and MALDI technique. For the purpose of relative quantification, stable isotopes can be introduced into the analytes during cellular protein synthesis,9,10 during protein digestion,11,12 or by derivatization of proteolytic peptides.13-15 Absolute quantifications can be performed by the addition of calibrated solutions of stable isotope labeled peptides as internal standards. These can be generated either by chemical synthesis16 or by the expression of a cleavable protein construct in a stable isotope labeled cell culture.17 As an alternative access, a labelfree assay for quantitative protein expression analysis that is based on the reproducibility of two independently performed proteome (5) Carr, S. A.; Huddleston, M. J.; Annan, R. S. Anal. Biochem. 1996, 239, 18092. (6) Neubauer, G.; Mann, M. Anal. Chem. 1999, 71, 235-42. (7) Peters, E. C.; Brock, A.; Ficarro, S. B. Mini Rev. Med. Chem. 2004, 4, 313324. (8) Lovet, K. M.; Stults, J. T.; Arnott, D. Mol. Cell. Proteomics 2005, 4, 235245. (9) Ong, S. E.; Blagoev, B.; Kratchmarova, I.; Kristensen, D. B.; Steen, H.; Pandey, A.; Mann, M. Mol. Cell. Proteomics 2002, 1, 376-386. (10) Pratt, J. M.; Robertson, D. H. L.; Gaskell, S. J.; Riba-Garcia, I.; Hubbard, S. J.; Sighu, K.; Oliver, S. G.; Butler, P.; Hayes, A.; Petty, J.; Beynon, R. J. Proteomics 2002, 2, 157-163. (11) Schno ¨lzer, M.; Jedrzejewski, P.; Lehmann, W. D. Electrophoresis 1996, 17, 945-953. (12) Bonenfant, D.; Schmelzle, T.; Jacinto, E.; Crespo, J. L.; Mini, T.; Hall, M. N.; Jenoe, P. Proc. Nat. Acad. Sci. U.S.A. 2003, 100, 880-885. (13) Gygi, S. P.; Rist, B.; Gerber, S. A.; Turecek, F.; Gelb, M. H.; Aebersold, R. Nat. Biotechnol. 1999, 17, 994-999. (14) Thompson, A.; Scha¨fer, J.; Kuhn, K.; Kienle, S.; Schwarz, J.; Schmidt, G.; Neumann, T.; Hamon, C. Anal. Chem. 2003, 75, 1895-1904. (15) Ross, P. L.; Huang, Y. L. N.; Marchese, J. N.; Williamson, B.; Parker, K.; Hattan, S.; Khainovski, N.; Pillai, S.; Dey, S.; Daniels, S.; Purkayastha, S.; Juhasz, P.; Martin, S.; Bartlet-Jones, M.; He, F.; Jacobson, A.; Pappin, D. J. Mol. Cell Proteomics 2004, 12, 1154-1169. (16) Gerber, S. A.; Rush, J.; Stemman, O.; Kirschner, M. W.; Gygi, S. P. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 6940-6945. (17) Beynon, R. J.; Doherty, M. K.; Pratt, J. M.; Gaskell, S. J. Nat. Methods 2005, 2, 587-589.

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analyses has been demonstrated.18 As a special variant of labelfree quantification in protein phosphorylation analysis, the determination of LC-ESI response factors within peptide/phosphopeptide pairs has been demonstrated by analysis of sample pairs interlinked by a kinase or phosphatase action.19 In recent years, our group has introduced phosphorus detection by element mass spectrometry (ICPMS, inductively coupled plasma mass spectrometry) for analysis of protein phosphorylation.20 This approach is highly specific for detection of phosphate groups at serine, threonine, and tyrosine (phosphoesters) or histidine (phosphoamide) introduced as posttranslational modification, since proteinogenic amino acids do not contain phosphorus. In addition, only few other, less common modifications introduce phosphorus into proteins (e.g., via a glycosylinositol phosphate anchor). The inherent advantage of this novel approach is that the ICP ionization efficiency of an element does not depend on its chemical form, making ICPMS a perfectly complementary technique to the molecular ionization techniques ESI and MALDI.21 Following the first report on phosphopeptide spotting by capillary liquid chromatography (µLC)-ICPMS with 31P monitoring,20 other applications in this field followed.22-25 In quantitative protein analysis by element MS, sulfur is a key element,26 since the sulfur-containing amino acids cysteine and methionine are embodied in the broad majority of proteins. By combined monitoring of phosphorus and sulfur, the protein phosphorylation stoichiometry can be assessed, which was demonstrated using µLCICPMS.27 Likewise, the metal-to-sulfur ratio can provide a measure for a metal/protein ratio in metalloproteins28 or a metal/peptide stoichiometry in chelator-tagged peptide constructs.29 The most established method for protein separation is sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) because of its high separation power. This technique has created a demand for further characterization of gel-separated proteins by chemical, biochemical, or spectroscopic techniques, which is an enduring challenge. Currently, the most widespread approach applies in-gel digestion with subsequent peptide analysis by mass spectrometry.30-32 Direct MS analysis of electroblotted, intact (18) Silva, J. C.; Denny, R.; Dorschel, C. A.; Gorenstein, M.; Kass, I. J.; Li, G. Z.; McKenna, T.; Nold, M. J.; Richardson, K.; Young, P.; Geromanos, S. Anal. Chem. 2005, 77, 2187-2200. (19) Steen, H.; Jebanathirajah, J. A.; Springer, M.; Kirschner, M. W. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 3948-3953. (20) Wind, M.; Edler, M.; Jakubowski, N.; Linscheid, M.; Wesch, H.; Lehmann, W. D. Anal. Chem. 2001, 73, 29-35. (21) Wind, M.; Lehmann, W. D. J. Anal. At. Spectrom. 2004, 19, 20-25. (22) Axelsson, B.-O.; Jo¨rnten-Karlsson, M.; Michelsen, P.; Abou-Shakra, F. Rapid Commun. Mass Spectrom. 2001, 15, 375-385. (23) Wind, M.; Kelm, O.; Nigg, E. A.; Lehmann, W. D. Proteomics 2002, 2, 15161523. (24) Wind, M.; Gosenca, D.; Ku ¨ bler, D.; Lehmann, W. D. Anal. Biochem. 2003, 317, 26-33. (25) Pro¨frock, D.; Leonhard, P.; Ruck, W.; Prange, A. Anal. Bioanal. Chem. 2005, 381, 194-204. (26) Wind, M.; Wegener, A.; Eisenmenger, A.; Kellner, R.; Lehmann, W. D. Angew. Chem., Int. Ed. 2003, 42, 3425-3427. (27) Wind, M.; Wesch, H.; Lehmann, W. D. Anal. Chem. 2001, 73, 3006-3020. (28) Hann, S.; Koellensperger, G.; Obinger, C.; Furtmu ¨ ller, P. G.; Stingeder, G. J. Anal. At. Spectrom. 2004, 19, 74-79. (29) Kru ¨ ger, R.; Braun, K.; Pipkorn, R.; Lehmann, W. D. J. Anal. At. Spectrom. 2004, 19, 852-857. (30) Henzel, W. J.; Billeci, T. M.; Stults, J. T.; Wong, S. C.; Grimley, C.; Watanabe, C. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 5011-5015. (31) Mann, M.; Wilm, M. Anal. Chem. 1994, 66, 4390-4399. (32) Henzel, W. J.; Watanabe, C.; Stults, J. T. J. Am. Soc. Mass Spectrom. 2003, 14, 931-942.

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proteins by MALDI-MS33-35 has been reported but has not found broad acceptance. In extension of previous work on application of ICPMS to phosphoprotein analysis, we have recently explored the application of ICPMS to direct analysis of blotted phosphoproteins.36 Upon scanning the blot by a laser, the ablated material was introduced into an ICPMS, and a 31P image was generated (imLA-ICPMS). Gels can also be analyzed directly by imLAICPMS;37,38 however, blots have lower volumes, offer a higher surface density of the analytes, and can be washed easily. We have extended the analysis of protein blots to simultaneous monitoring of P and S. Comparison of the two element-specific images allows the detection of protein bands based on the S intensities as well as specific spotting of phosphorylated proteins based on the P intensities. In contrast to most common staining techniques, the pictures are quantitative by nature, and phosphorylation stoichiometries of undigested proteins can be measured. In this study, we demonstrate that either imLA-ICPMS or µLCICPMS gives easy and reliable access to quantitative phosphorylation stoichiometry data. Furthermore, we extend the ICP-based approach to the analysis of subproteomes. EXPERIMENTAL SECTION Protein Preparation. Cells from Corynebacterium glutamicum were pelleted and disrupted using a French press in the presence of a detergent solution containing a mixture of phosphatase and protease inhibitors (phosphosafe, Novagen) and DNAse I, followed by ultracentrifugation and acetone precipitation. Cultured mouse (Mus musculus) fibroblasts were lysed directly by addition of phosphosafe buffer including DNAse I to the cells. Simultaneous protein precipitation and phospholipid depletion were achieved by addition of ethanol. After protein precipitation overnight, the samples from both cell types were pelleted; air-dried; and finally, redissolved in detergent-containing buffer. Protein concentrations were determined by the Quick Lowry Assay (Biorad). Gel Electrophoresis and Protein Digestion. SDS-PAGE and protein blotting were performed according to standard procedures. One-dimensional SDS-PAGE was accomplished with commercially available precast gels (NuPAGE gels, Invitrogen) using potentiostatic conditions (200 V). For determination of the phosphorylation stoichiometry of soluble proteins, 50 µg of total protein lysate was applied per gel lane. The migration distance was restricted to 2-3 cm (10-15 min). Proteins were blotted onto PVDF membranes at 300 mA for 45 min using a transfer buffer consisting of 25 mM Tris, 192 mM glycine buffer, and 20% methanol. Reduction with DTT (45 min at 54 °C) and alkylation with iodoacteamide (30 min at room temperature) was applied prior to digestion. Proteins were digested in-gel at 37 °C overnight using modified trypsin (Roche). Alternatively, a combination of AspN and trypsin was used. Proteolytic peptides were extracted (33) Eckerskorn, C.; Lottspeich, F. Electrophoresis 1990, 11, 554-561. (34) Eckerskorn, C.; Strupat, K.; Schleuder, D.; Hochstrasser, D.; Sanchez, J. C.; Lottspeich, F.; Hillenkamp, F. Anal. Chem. 1997, 69, 2888-2892. (35) Schreiner, M.; Strupat, K.; Lottspeich, F.; Eckerskorn, C. Electrophoresis 1996, 17, 954-961. (36) Wind, M.; Feldmann, I.; Jakubowski, N.; Lehmann, W. D. Electrophoresis 2003, 24, 1276-1280. (37) Marshall, P.; Heudi, O.; Bains, S.; Freeman, H. N.; Abou-Shakra, F.; Reardon, K. Analyst 2002, 127, 459-461. (38) Ma, R. L.; McLeod, C. W.; Tomlinson, K.; Poole, R. K. Electrophoresis 2004, 25, 2469-2477.

2-3 times with 5% formic acid and desalted with RP-C18 micropipet tips (ZipTip, Millipore). Capillary LC. Capillary liquid chromatography was performed with a dual syringe pump (ABI 140B, Applied Biosystems). A reversed-phase C18 column (Grohm/Vydac) of the following dimensions was used: 200-µm i.d., 15-cm length, 5-µm particle size. The peptides were separated by a linear gradient from 2% acetonitrile/0.065% TFA to 80% acetonitrile/0.1% TFA within 30 min. The LC column outlet was directly coupled to the ICPMS via a microconcentric nebulizer with a low-volume spray chamber as interface (CEI-100, CETAC). Laser Ablation. Blot membranes were analyzed by LA-ICPMS using a Nd:YAG laser emitting at λ ) 213 nm (UP-213, New Wave) using argon as carrier gas. The laser ablation unit was equipped with a sample chamber with minimized dead volume and optimized flow characteristics (SuperCell, New Wave) and connected directly to the injector of the ICPMS. The following laser parameters were used: laser energy, 0.15 mJ; frequency, 10 Hz; and focus, 150 µm. Parallel line scans of the blots were performed in direction of electrophoretic migration with a feed rate of 150 µm/s, and the sum of these traces was used for quantitative evaluation. ICPMS. A double-focusing sector-field instrument (Element 2, Thermo Electron) at a resolution of 4000 (medium resolution) was used. The instrument was tuned via infusion of a multielement standard with a syringe pump. The following conditions were found to be optimal: sample gas flow, 1.2-1.3 L/min; auxiliary gas flow, 0.6-0.8 L/min; cool gas flow, 16 L/min; and RF power, 1250-1350 W. To achieve high repetition rates and high duty cycles for detection of transient signals, peak tops (20% mass window) were monitored using the autolockmass feature. The electric scan mode was used exclusively to avoid long magnet settling times. The resulting overall scan cycle time was 200400 ms per run for monitoring 31P and 34S with a duty cycle of nearly 50% per element. Data evaluation was performed after ASCII export to the graphics software Origin version 6.1G. RESULTS AND DISCUSSION General Strategy. The major goal of this study was to evaluate element mass spectrometry for analysis of the proteomic phosphorylation degree (or phosphorylation stoichiometry). The extent of phosphorylation is considered to be essential for phosphoprotein function, since it may determine, for example, its enzymatic activity, its subcellular localization, or its interaction with partners in a signaling cascade. As outlined above, current techniques for determination of the protein phosphorylation degree are complex. We pursue a robust and straightforward novel attempt for this purpose based on element mass spectrometry. This methodology does not require the introduction of a label or sample derivatization and provides analytical data, from which the desired quantitative information is easily extracted. We employed the two strategies schematically outlined in Figure 1, aiming at proteolytic peptides of phosphoproteins (Figure 1a) and intact phosphoproteins (Figure 1b), respectively. In the LC/MS approach, proteins were digested in-gel, and the proteolytic peptide mixture was subjected to reversed-phase capillary LC-ICPMS (Figure 1a). The on-line coupling of µLC to ICPMS was achieved using a miniaturized capillary-based interface

Figure 1. Schematic display of the two strategies employed in the study of protein phosphorylation analysis: (a) proteins are separated by 1D-PAGE, digested in-gel, and the peptides are analyzed by capillary LC-ICPMS; (b) proteins are separated by 1D-PAGE and blotted onto PVDF membranes, which are subjected to imaging LAICPMS.

designated for low flow rates and low dead volumes.39 In the LAICP approach (Figure 1b), intact phosphoproteins were analyzed directly by laser ablation from blot membranes. The blot membranes were ablated with parallel line scans, and the data were then converted into color-coded 2D-images (imaging LA-ICPMS). In both approaches, element-specific traces of 31P+ and 34S+ were monitored simultaneously. Detection of phosphorus and sulfur with ICPMS is compromised by isobaric polyatomic interferences. To exclude these interferences, a sector-field mass analyzer was applied. Medium resolution (R ) 4000) is sufficient to achieve baseline separation of the nitrogen- and oxygen-based interferences from 31P+ and 34S+, where the closest interfering signals (NO+, O2+) appear at a distance of 18 and 26 mDa, respectively, on their high-mass side. For detection of S, 34S+ was preferred over 32S+, since a slightly better analytical performance was obtained. Analysis of Enzymatic Protein Digests by Capillary LCICPMS. Sulfur is embodied in the broad majority of proteins via the amino acids cysteine and methionine. Thus, sulfur can be used as the internal reference for relative quantification of phosphorus in sulfur-containing proteins. The reliability of this LC approach for determination of the phosphorylation stoichiometry has been shown for intact phosphoproteins.27 Once the relative response factors for 31P and 34S have been determined for a particular LC separation, these factors can be applied for all subsequent analyses by this method. ICP sensitivity factors for 31P and 34S as a function of the LC solvent composition were determined for the binary solvent system A (0.065% TFA) and B (0.1% TFA/80% acetonitrile) using calibration solutions with phosphoserine and cysteine. From the sensitivity factors determined for different eluent ratios, a continuous sensitivity correction function was calculated. This function (see Figure 2) was then used for correction of the sulfur signal intensities. The application of this correction function for a µLCICPMS analysis is shown in Figure 2. Chicken ovalbumin was digested by a mixture of trypsin/AspN, and the proteolytic peptides were analyzed by reversed-phase µLC-ICPMS. Several intense peaks in the 31P trace indicate the elution of phosphopeptides, whereas the peaks on the 34S trace represent cysteine- or methionine-containing peptides. Taking into account the relative natural abundance of 34S of 4.29%, a P/S ratio can be (39) Schaumlo ¨ffel, D.; Ruiz Encicar, J.; Lobinski, R. Anal. Chem. 2003, 75, 68376842.

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Figure 2. µLC-ICPMS chromatogram of an ovalbumin in-gel digest (trypsin/AspN). 31P (blue) and 34S (red) were monitored simultaneously. The 31P/34S sensitivity correction function (see text) is shown in black. Peaks on the 31P trace indicate the elution of phosphopeptides; signals on the 34S trace represent peptides with cysteine, methionine, or both. Integration of all peaks on the respective traces gives access to a P/S ratio representing the protein phosphorylation stoichiometry. The experimental result for ovalbumin was 0.080 mol of P/mol of S or 1.76 mol of P/mol of protein (22 sulfur atoms per protein).

calculated from the sum of the peak integrals of the two traces, which resembles the phosphorylation stoichiometry of the analyzed protein. From the data in Figure 2, a P/S ratio of 0.080 mol of P/mol of S was calculated for ovalbumin. A similar result was obtained upon analysis of intact ovalbumin by µLC-ICPMS, in which a P/S-ratio of 0.064 mol of P/mol of S was measured. The knowledge of the number of S-containing amino acids in ovalbumin (22 ) 6 Cys + 16 Met) allows the calculation of a proteinic phosphorylation degree mol of P/mol of protein. For ovalbumin, a value of 0.080 mol of P/mol of S corresponds to 1.76 mol of P/mol of protein. This value is very close to a literature value of 1.73 mol of P/mol of protein obtained by the malachite-green method.40 This demonstrates that determination of the protein phosphorylation stoichiometry is feasible via analysis of a protein digest. Sample preparation steps, such as reduction, alkylation, digestion, and peptide extraction may influence the P/S ratio determined by µLC-ICPMS; however, our first quantitative results with this analytical strategy look promising. We applied phosphatase-catalyzed dephosphorylation41 to check that the phosphorus peaks shown in Figure 2 originated from phosphopeptides. For this purpose, one aliquot of ovalbumin was incubated with alkaline phosphatase for 1 h. Both this sample and an untreated sample were digested, desalted, and analyzed by µLC-ICPMS. The highly abundant peaks on the 31P trace in the untreated sample are almost completely eliminated upon enzymatic dephosphorylation (Figure 3, lower trace), whereas the 34S trace essentially remained unaltered by this treatment (Figure 2, upper trace). This result confirms that the 31P signals in the untreated sample are caused by phosphomonoesters. For application of the proposed strategy, interference from other P- or S-containing compounds, such as (oligo)-nucleotides, phospholipids, or small inorganic molecules such as phosphate (40) Ekman, P.; Ja¨ger, O. Anal. Biochem. 1993, 214, 138-141. (41) Larsen, M. R.; Sorensen, G. L.; Fey, S. J.; Larsen, P. M.; Roepstorff, P. Proteomics 2001, 1, 223-238.

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Figure 3. Comparison of µLC-ICPMS chromatograms of ovalbumin in-gel digests (trypsin) without and with incubation with alkaline phosphatase. The two 34S traces are practically identical (black, without; red, with phosphatase treatment). Three major peaks are visible in the 31P trace before phosphatase treatment (lower panel, blue), which are almost absent after incubation (lower panel, green). Thus, the occurrence of phosphomonoesters is confirmed.

or sulfate, has to be excluded. In our analytical strategy, this is achieved by several purification steps, including SDS-PAGE, desalting, and LC. As a result, the element-specific traces obtained from protein digests are highly reproducible (see Figure 3, upper panel). The method is also suited to characterize the efficiency of the digestion procedure. By comparison of the total sulfur intensities before and after enzymatic digestion, the extraction efficiency from the gel can be estimated relative to the applied protein amount. In the example shown in Figure 2, 17% of the original sulfur intensity was found after in-gel digestion using a trypsin/AspN mixture. This recovery was significantly higher than that achieved by using trypsin alone (9% recovery, Figure 3). Possibly, the smaller average size of the peptides generated by the use of trypsin/AspN, as compared to trypsin, leads to higher recovery. This example shows that the peptide extraction efficiency may be improved by the use of enzyme mixtures. Imaging LA-ICPMS of Protein Blots. The LC-ICPMS strategy described in the previous section relies on enzymatic cleavage and subsequent peptide extraction. Although this approach currently offers the lowest detection limits, the numerous sample preparation steps may introduce errors in quantifications. These may be reduced by analysis of intact proteins. Although LC is increasingly applied to protein separation, it is not as straightforward as LC of peptides. In size-exclusion chromatography (SEC), proteins show broad peaks, poor separation, and a tendency to form nonspecific adducts, but standard reversed-phase HPLC has limited separation efficiency for proteins, as well. Therefore, the protein separation technique of choice is currently SDS-PAGE, in which high separation efficiencies can be achieved. Proteins are usually visualized in-gel by staining techniques. For further analysis, such as antibody staining, proteins are often blotted onto membranes by electroelution perpendicular to the separation axis. By this way, proteins can be trapped on nitrocellulose or PVDF membranes. Thus, the major part of the proteins is concentrated on the blot surface, which makes protein blots ideal targets for further chemical or spectroscopic characterization.

Figure 4. Separation of a mixture of 4 proteins (lactoperoxidase, ovalbumin, R-casein, myoglobin; 10, 20, 20, 20 pmol, respectively) with 1D SDS-PAGE. (a) Gel separation (schematic); measures for the P and S content of the proteins used are indicated on the left side. (b) imLAICPMS: 31P analysis of the PVDF-blot; (c) imLA-ICPMS: 34S analysis of the PVDF-blot. 31P and 34S show detection limits of 30 pmol and 8 pmol, respectively. Eight picomoles of 34S correspond to ∼200 pmol of total sulfur. Using the 31P/34S ratio, the phosphorylation degree of ovalbumin and R-casein can be calculated. For ovalbumin, 1.9 mol of P/mol of protein and for R-casein, 7.8 mol of P/mol of protein was measured.

In a pilot study, imaging laser ablation ICPMS (imLA-ICPMS) with 31P detection was applied to the analysis of phosphoproteins blotted onto PVDF membranes.36 In the present study, we extended this approach to simultaneous monitoring of 31P and 34S with the intention of determining the protein phosphorylation degree in the same way as demonstrated above using µLCICPMS analysis of phosphoprotein digests. The surface of the blot membrane is scanned line-by-line (see Figure 1b), and the element-specific traces can be combined to element-specific images, which are equal to results obtained by staining techniques. Figure 4 shows 31P and 34S images of a protein blot where a standard protein mixture composed of two phosphorylated and two nonphosphorylated proteins was separated by 1D-SDSPAGE, blotted onto a PVDF membrane, and analyzed by imLAICPMS. One intense and one faint band are visible in the 31P image (Figure 4b), indicating that the corresponding proteins are phosphorylated. Two intense and one very faint band appear on the 34S image; myoglobin, with only two S per molecule is not visible. For the two phosphoproteins, the phosphorylation stoichiometry can be calculated from the integral of their 31P and 34S signals. In contrast to a gradient LC analysis, the sensitivity of 31P and 34S detection with imLA-ICPMS is constant over the whole blot. The sensitivity factor P/S for the imLA analysis was determined to be 0.6 by incubation of the blot membrane with phosphoserine and cysteine, and all raw data intensities were corrected by this factor. The experimental P stoichiometries (sum of all traces) for the analysis shown in Figure 4 were 1.9 mol of P/mol of protein for ovalbumin and 7.8 mol of P/mol of protein for R-casein, respectively. The result for ovalbumin is close to that obtained by µLC-ICPMS following enzymatic in-gel digestion, and the value obtained for R-casein is in accordance with its about eight completely phosphorylated sites, as determined by ESI-MS.27 One inherent advantage of the imLA-based approach is that the analysis is performed on the level of intact proteins and that no acidic pH is applied during sample preparation and analysis. This opens the potential to detect acid-labile compounds or

modifications. Another advantage is the visualization of the element information, which allows an easy and direct comparison with staining techniques. By using the line-scan approach in combination with the generation of element-specific images, protein bands can be identified safely, and background subtraction can be accomplished easily. The recognition of true analyte signals is not as straightforward in the case a discontinuous ablation of selected gel spots is performed. In general, the analysis of protein blots, as compared to gel analysis, leads to improved purification, concentration of the proteins at the surface, and lower blank values. Comparison of µLC-ICPMS and imLA-ICPMS. The phosphorylation degree data determined by µLC-ICPMS and imLAICPMS show a precision of 20-40% employing relative quantification. All in all, both presented methods allow a reliable determination of the protein phosphorylation degree. Table 1 gives a method comparison, where some figures of merit are depicted for the two presented approaches. For calculation of the detection limits given in Table 1, all sample preparation steps were considered. If no purification is needed, LC-only methods are fast, and µLC offers the lowest detection limits, but this approach is restricted to isolated proteins. Methods which combine electrophoretic separation with a second purification step (µLC or protein blotting) allow the analysis of more complex samples, but they are compromised by higher limits of detection. This difference is mainly related to the sample preparation, for example, analyte loss upon peptide extraction from the gel or incomplete blotting. Moreover, the overall signal intensity may be split into several peaks upon digestion and LC of the proteolytic peptides. Dilution of signal intensity is also valid in the case of gel bands, which may cover a significant area on the blot (1 × 4 mm). Acid-catalyzed alterations may be avoided by SEC-ICPMS or imLA-ICPMS analysis from protein blots. In principle, SEC-ICPMS allows analysis under nondenaturing conditions; however, great care must be taken to avoid nonspecific adducts, in particular, protein/protein and protein/phospholipid adducts. Analytical Chemistry, Vol. 78, No. 6, March 15, 2006

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Table 1. Figures of Merit for Determination of the Protein Phosphorylation Stoichiometry, Including the Two Proposed Strategies Suited for Analysis of Crude Protein Mixturesa limit of detection (LOD)b

SEC rp-µLC gel electrophoresis + digest + rp-µLC gel electrophoresis + protein blot + laser ablation

P/protein, pmol

S/protein, pmol

analysis time, h

purification efficiency

acidic conditions

protein denaturation

10 0.2-0.5 1-5 30

200 10-25 200-400 200

1 1 8-24 8

low medium high high

no yes yes no

no yes peptides yes

a LC-only methods (size exclusion chromatography (SEC), reversed-phase (rp)-µLC) exhibit lower detection limits, but are restricted to isolated proteins. Data refer to the complete analytical procedure, including sample preparation. b LOD may vary depending on analyte recovery, retention time etc.

Determination of the Proteomic Phosphorylation Degree. As extension of the analyses described above, we examined protein samples of higher complexity. The phosphorylation status of a cellular or subcellular proteome is of interest, since it can reflect the cell type or its actual functional status.42 Despite the enormous efforts for proteome-wide analysis and comparison of different cell states, a measure of the native phosphorylation status of a proteome currently cannot be obtained easily. In a first attempt in this direction, we applied the ICPMS-based methods to the soluble proteome fractions of C. glutamicum and M. musculus (mouse 3T3 cells). Corresponding cells were lysed, and the proteins were precipitated in the presence of phosphatase and protease inhibitors. DNA was cleaved into single nucleotides by addition of DNAse, and the majority of phospholipids was extracted by an ethanol extraction step. A major issue for analysis of crude protein fractions for phosphoproteins by analysis of the elements P and S is the exclusion of compound classes such as phospholipids; nucleotides; or small molecules, such as phosphate, sulfate, and SDS. The two-step purification/separation scheme as applied in the two ICPMS approaches presented above appears to provide a good separation of proteins from these interfering compounds. Key principles of sample cleanup are the 1D-gel electrophoresis step combined either with digestion, peptide extraction, and µLC (in the µLC-ICPMS strategy) or the use of protein blotting (in the imLA-ICPMS strategy). Nucleotides and phospholipids are removed by gel electrophoresis, since they concentrate at the migration front. Buffer components such as SDS are removed either during workup of the gel bands or by the blotting step. Figure 5 shows the ICPMS results for two different proteomes, one derived from a prokaryotic (C. glutamicum) and one derived from a eukaryotic (M. musculus) cell culture. Both proteomes were analyzed by µLC-ICPMS and imLA-ICPMS as described above. In the chromatograms of the digest samples, intense S signals indicate the elution of peptides (see Figure 5a,b, red traces). Whereas the 31P signals (Figure 5a,b, blue traces) are of equal height in the case of the mouse proteome digest, there are only a few weak signals in the 31P-trace of the corresponding sample from C. glutamicum. This result obtained for C. glutamicum is in accordance with the low number of protein kinase genes in prokaryotes in general (42) Bandura, D. R.; Ornatsky, O. I.; Liao, L. J. Anal. At. Spectrom. 2004, 19, 96-100.

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and in the genome of C. glutamicum in particular.43 Since acidic conditions were applied for peptide extraction from the gel and for LC, acid-labile phosphorylation sites, such as phosphohistidine or phosphoaspartate, which play a major role in prokaryotic cell signaling, will probably not survive sample preparation. Thus, the low abundance of the 31P signal reflects the low abundance of phosphoserine, phosphothreonine, and phosphotyrosine in the C. glutamicum proteome. In agreement with the more prominent role of serine, threonine, and tyrosine kinases in eukaryotes, the 31P trace obtained in the case of the protein digest from mouse cells (Figure 5b) showed much higher intensities, indicating the elution of a higher number of phosphopeptides. The calculated molar P/S ratio was 0.032 for mouse cells, which is ∼30 times higher as compared to the molar P/S ratio of 0.001 calculated for C. glutamicum. The images obtained by imLA-ICPMS also show this difference very clearly: The blot from the mouse proteins (Figure 5f) shows abundant 31P spots, whereas the blot from the bacterial proteins (Figure 5e) shows almost no 31P signals. In both cases, the 34S image resembles the picture obtained by Coomassie blue staining of a duplicate sample and, thus, the spatial protein distribution (Figure 5c,d). The overall low 31P intensity in the case of C. glutamicum (see Figure 5e) prohibits a clear assignment of P signals to phosphoproteins and the calculation of a P/S ratio, which reflects also the higher detection limit of imLA-ICPMS in comparison to µLC-ICPMS. In contrast, the 31P image in the case of the mouse cells reveals a distinct distribution of phosphoproteins with much higher 31P intensity, similar to the 34S image (Figure 5f). For mouse cells, the molar P/S ratio was calculated at 0.036, which is slightly higher than the value obtained upon enzymatic digestion and µLC-ICPMS of proteolytic peptides. One could speculate that acid labile phosphorylation sites do have a higher chance to survive sample preparation in the case of protein blots, since no acidic step is involved. On the other hand, the low 31P abundance observed in the prokaryotic proteome may indicate that labile phosphorylation sites are not completely preserved. The results of the two methods confirm each other and are in agreement with other biological information, for example, from the genome structure. The molar P/S ratios determined for the (43) Kalinowski, J.; Bathe, B.; Bartels, D.; Bischoff, N.; Bott, M.; Burkovski, A.; Dusch, N.; Eggeling, L.; Eikmanns, B.; Gaigalat, L.; Goesmann, A.; Hartmann, M.; Huthmacher, K.; Kra¨mer, R.; Linke, B.; McHardy, A. C.; Meyer, F.; Mo ¨ckel, B.; Pfefferle, W.; Pu ¨ hler, A.; Rey, D. A.; Ru ¨ ckert, C.; Rupp, O.; Sahm, H.; Wendisch, V. F.; Wiegra¨be, I.; Tauch, A. J. Biotechnol. 2003, 104, 5-25.

Figure 5. Phosphorylation analysis of proteomes from C. glutamicum and M. musculus by the two ICPMS-based approaches. (c, d) Cells were lysed, and the proteins were precipitated. The extracted soluble protein fraction was then subjected to a short (2-3 cm) 1D-SDS-PAGE intended for protein purification. After gel electrophoresis, the samples were analyzed by two methods: (a, b) in-gel digestion with trypsin and analysis by µLC-ICPMS or (e, f) proteins were blotted without staining onto a PVDF membrane, and analysis was performed by imLA-ICPMS. Both techniques reveal that protein phosphorylation is significantly more abundant in the case of the eukaryotic cells ( M. musculus), as compared to the bacterial cells (C. glutamicum).

subproteomes can be converted into a subproteomic phosphorylation stoichiometry mol of P/mol of protein; this conversion is performed in the same way as for data from individual proteins. The required data, proteomic abundance of cysteine and methionine and the average protein molecular weight, have to be inferred from the genome organization. It was assumed that the encoded average amino acid abundance is reflected in the expressed proteome. For the protein molecular weight, an average value of 50 kDa was assumed for both cases (mouse and bacterium). This corresponds to 395 amino acids/protein for C. glutamicum and

449 amino acids/protein for mouse cells, calculated using the corresponding average amino acid molecular weights.44,45 For C. glutamicum, the abundance of cysteine and methionine amounts to 2.94% on average,44 which equals 11 sulfur atoms per protein. In the same way, an average abundance of cysteine and methionine of 4.59% in mouse cells45 is equivalent to 21 sulfur atoms per (44) Nishio, Y.; Nakamura, Y.; Kawarabayasi, Y.; Usuda, Y.; Kimura, E.; Sugimoto, S.; Matsui, K.; Yamagishi, A.; Kikuchi, H.; Ikeo, K.; Gojobori, T. Genome Res. 2003, 13 (7), 1572-1579. (45) Xia, X.; Xie, Z. Mol. Biol. Evol. 2002, 19, 58-67.

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protein. Thus, the experimental P/S ratio of 0.001 for C. glutamicum is equivalent to a P stoichiometry of 0.012 mol of P/mol of protein (see Figure 5a). In a simple picture, this is equivalent to ∼1% of the proteins being phosphorylated at one site to 100%. Such a low phosphorylation degree is in rough accordance with antibody- and 33P-labeling data.46 It was concluded that ∼3% of the proteins in C. glutamicum are phosphorylated at least at one site with an undefined degree of phosphorylation. For mouse cells, the experimental molar P/S ratio of 0.032 is equivalent to a P stoichiometry of 0.665 mol of P/mol of protein in the case of the µLC-ICPMS method (see Figure 5b), whereas the experimental molar P/S ratio of 0.036 is equivalent to a slightly higher P stoichiometry of 0.87 mol of P/mol of protein in the case of the imLA-ICPMS method (see Figure 5f). This value is equivalent to ∼2/3 of the soluble mouse proteins being completely phosphorylated at one site. In reality, the proteins occur as a mixture of nonphosphorylated, highly (multiply) phosphorylated, and weakly phosphorylated proteins, a situation of remarkable structural heterogeneity that remains to be analyzed. CONCLUSIONS We have introduced and demonstrated the use of element mass spectrometry for the determination of the degree of phosphory(46) Bendt, A. K.; Burkovski, A.; Schaffer, S.; Bott, M.; Farwick, M.; Hermann, T. Proteomics 2003, 3, 1637-1646.

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lation on the protein and proteome level by a label-free and derivatization-free procedure. This enables for the first time a direct and fast deduction of a global phosphorylation degree for a complex protein mixture. The methods also provide new access to the native phosphorylation status of cell-type-specific proteomes or of subcellular proteomes. Solid evidence has accumulated that numerous proteins involved in cellular proliferation exhibit an elevated degree of phosphorylation in mitosis, as compared to their phosphorylation status in resting cells.2 A first hint that a global phosphorylation degree may contain biological information has been demonstrated in the investigation of malignant cells42 with possible application to cancer diagnosis and prognosis. ACKNOWLEDGMENT We are indebted to Dirk Schaumlo¨ffel, Pau, as well as to Christopher Latkozcy and Detlef Gu¨nther, Zu¨rich, for valuable support. Financial support of the Bundesministerium fu¨r Bildung und Forschung (BMBF, Proteomics Program) is gratefully acknowledged.

Received for review October 24, 2005. Accepted January 3, 2006. AC051896Z