Anal. Chem. 2001, 73, 3006-3010
Protein Phosphorylation Degree: Determination by Capillary Liquid Chromatography and Inductively Coupled Plasma Mass Spectrometry Mathias Wind,† Horst Wesch,‡ and Wolf D. Lehmann*,†
Central Spectroscopy and Department of Biophysics, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany
Capillary liquid chromatography (µLC) interfaced to inductively coupled plasma mass spectrometry (ICPMS) is introduced as a new micromethod to determine the phosphorylation degree in phosphoproteins and phosphopeptides containing cysteine and/or methionine residues. The stoichiometric phosphorus to sulfur (31P to 32S) ratio is experimentally determined by µLC-ICPMS and converted into the degree of phosphorylation using protein/ peptide sequence information. The method is applied to the phosphoproteins r-casein, β-casein, and recombinant protein kinase A catalytic subunit and to synthetic phosphopeptides. The accurate data obtained by µLC-ICPMS allow quantitative assessment of the compound-specific discrimination of the electrospray ionization process between nonphosphorylated and phosphorylated proteins and peptides. Phosphorylation is probably the most abundant and biologically most important covalent modification of proteins.1,2 Kinasecatalyzed phosphorylation and phosphatase-catalyzed dephosphorylation are the two major antagonizing principles acting, for example, in short-term regulation of metabolic or signal transduction pathways. Often one form (phospho or dephospho state, respectively) represents the active state, whereas the counterpart is inactive. Despite of the biological significance of the protein phosphorylation degree, there is no standard method available for its determination. This is because current microanalytical methods in general do not measure a common property of both the nonphosphorylated and the phosphorylated form of a protein or peptide. Instead, methods normally are optimized toward exclusive detection of the phosphorylated form. For instance, the standard 32P/33P technology measures the incorporation of radioactive phosphorus, for example, after addition of radiolabeled ATP, GTP, or inorganic phosphate.1 This approach has also been used in combination with mass spectrometry to quantitate the amount * Corresponding author: (tel) 0049-6221-424563; (fax) 0049-6221-424554; (email)
[email protected]. † Central Spectroscopy. ‡ Department of Biophysics. (1) Protein Phosphorylation, Parts A and B; Hunter, T., Sefton, B. M., Eds.; Methods in Enzymology Vols. 200 and 201; Academic Press: San Diego, CA, 1991. (2) Protein Phosphorylation; Marks, F., Ed.; VCH: Weinheim, 1996.
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of phosphopeptides produced by in vitro phosphorylation.3 The use of anti-phosphopeptide antibodies is another frequently used methodology, and here the nonphosphorylated counterpart also escapes detection. However, this limitation may be overcome by use of two antibodies, one being directed against the phosphorylated, the other against the nonphosphorylated form.4 The analysis of different protein phosphorylation states has been demonstrated by the use of whole-cell stable isotope labeling and subsequent mass spectrometry.5 Another approach for determination of the degree of phosphorylation consists of a separate quantification of a phosphoprotein and of covalently bound phosphate following its hydrolytic release.6,7 Mass spectrometry (MS) in principle is capable of measuring the phosphorylation degree as a single method, since both the phospho and the dephospho forms of a protein (or proteolytic peptide) are measured by a common property, their mass. However, both electrospray ionization (ESI)8 and matrix-assisted laser desorption/ ionization (MALDI)9 show compound-specific ionization efficiencies. Using the standard positive ion mode, this results in reduced ionization efficiencies for phosphopeptides compared to their nonphosphorylated counterparts for both ESI10-12 and MALDI.13,14 The discriminatory effect increases with increasing number of phosphate residues present. Thus, estimation of the phosphoryl(3) Goodlett, D. R.; Aebersold, R.; Watts, J. D. Rapid Commun. Mass Spectrom. 2000, 14, 344-348. (4) Vanmechelen, E.; Vanderstichele, H.; Davidsson, P.; Van Kerschaver, E.; Van der Perre, B.; Sjogren, M.; Andreasen, N.; Blennow, K. Neurosci. Lett. 2000, 285, 49-52. (5) Oda, Y.; Huang, K.; Cross, F. R.; Cowbury, D.; Chait, B. T. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 6591-6596. (6) Ekdahl, K. N.; Ro ¨nnblom, L.; Sturfeld, G.; Nilsson, B. Arthritis Rheum. 1997, 40, 2178-2186. (7) Haglund, A. C.; Ronquist, G.; Frithz, G.; Ek, P. Thromb. Res. 2000, 98, 147-156. (8) Yamashita, M.; Fenn, J. B. J. Phys. Chem. 1984, 88, 4451-4459. (9) Karas, M.; Bachmann, D.; Bahr, U.; Hillenkamp, F. Int. J. Mass Spectrom. Ion Processes 1987, 78, 53-68. (10) Annan, R. S.; Huddleston, M. J.; Verma, R.; Deshaies, R. J.; Carr, S. A. Anal. Chem. 2001, 73, 393-404. (11) Carr, S. A.; Huddleston, M. J.; Annan, R. S. Anal. Biochem. 1996, 239, 180192. (12) Wind, M.; Edler, M.; Jakubowski, N.; Linscheid, M.; Wesch, H.; Lehmann, W. D. Anal. Chem. 2001, 73, 29-35. (13) Craig, A. G.; Hoeger, C. A.; Miller, C. L.; Goedken, T.; Rivier, J. E.; Fischer, W. H. Biol. Mass Spectrom. 1994, 23, 519-528. (14) Matsumoto, H.; Kahn, E. S.; Komori, N. Anal. Biochem. 1998, 260, 188194. 10.1021/ac010066s CCC: $20.00
© 2001 American Chemical Society Published on Web 05/24/2001
ation degree from positive ion ESI15 and MALDI mass spectral patterns results in false low values. We have solved the problem of accurately determining the extent of phosphorylation by applying inductively coupled plasma (ICP) mass spectrometry. This quantitative technique for element analysis has seen increasing biochemical applications in recent years, particularly when directly coupled to chromatography.16 These applications include detection of sulfur-containing anions,17 quantification of protein-bound metals,18 quantification of DNAadducts,19 and phosphopeptide detection.12 In the following, we introduce a new approach for accurate determination of the phosphorylation degree of proteins and peptides using capillary liquid chromatography (µLC)-ICPMS. EXPERIMENTAL SECTION Materials. Water and acetonitrile (ACN) were of HPLC grade from E. Merck (Darmstadt, Germany). Trizma-base, Ba(OH)2, and R- and β-casein were purchased from Sigma (St. Louis, MO). Bacterial alkaline phosphatase (activity, 0.3 unit/mg) was from Applichem (Darmstadt, Germany). Trifloroacetic acid (TFA) and all other chemicals were of analytical grade quality. Synthesis of phosphopeptides was performed by the F-moc technology in an automated peptide synthesizer AMS 422 (Abimed Analysentechnik). ZipTip desalting tips were purchased from Millipore (Bedford, MA). Sample Preparation. Peptide dephosphorylation was performed by addition of a few milligrams of solid Ba(OH)2 to a 10 µM aqueous peptide solution and subsequent stirring at room temperature for 4 h. Dephosphorylation of R-casein was performed with alkaline phosphatase. To a 50 µM R-casein solution in 0.2 M Tris-HCl buffer, pH 8, alkaline phosphatase was added to a concentration of 1.3 µM. The molar ratio substrate/enzyme was ∼40. The solution was incubated for ∼3 h at 37 °C. Immediately before analysis by nanoESI, peptide and protein samples were desalted using ZipTip microtips. Capillary LC. For HPLC a dual-syringe solvent delivery system (type 140B, Applied Biosystems, Foster City, CA) was used. Samples were injected using a 5-µL stainless steel sample loop. For separation, a Vydac C18 column (0.3 mm × 250 mm, 5 µm, 300 Å, LC Packings, Amsterdam, The Netherlands) was used. The standard gradient used was 0-5 min 5% B isocratic; 5-50 min 5-100% B linear. Mobile phase A was water/TFA 100:0.065 (v/ v) and B was ACN/water/TFA 80/20/0.05 (v/v/v). Solvents were degassed by helium. The total flow was 62 µL/min, and a split of 1:15 was used to achieve a flow of ∼4 µL/min over the column. For direct infusion experiments, a syringe pump type Harvard 01760 (Harvard Apparatus, Holliston, MA) was employed. LC-ICPMS. The ICP analyses were performed on a sector field mass spectrometer-type Element 2 (Thermoquest, Bremen, Germany) with medium mass resolution of 4000, which is sufficient for interference-free detection of 31P and 32S. A microflow (15) Graham, M. E.; Dickson, P. W.; Dunkley, P. R.; Nagy-Felsobuki, E. I. Anal. Biochem. 2000, 281, 98-104. (16) Sutton, K.; Sutton, R. M.; Caruso, J. A. J. Chromatogr., A 1997, 789, 85126. (17) Divjak, B.; Goessler, W. J. Chromatogr., A 1999, 844, 161-169. (18) Leber, A.; Hemmens, B.; Klo ¨sch, B.; Goessler, W.; Raber, G.; Mayer, B.; Schmidt, K. J. Biol. Chem. 1999, 274, 37658-37664. (19) Siethoff, C.; Feldmann, I.; Jakubowski, N.; Linscheid, M. J. Mass Spectrom. 1999, 34, 421-426.
Table 1. Accurate Masses Values of 31P, 32S, and the Most Abundant Interferring Molecular Ions Observed in ICPMS Brutto formula 31P 15N16O 14N16O1H 12C18O1H 32S 16O16O 14N18O 15N16O1H
accurate mass 30.97376 30.99502 31.00581 31.00699 31.97207 31.98982 32.00223 32.00285
nebulizer (Microflow PFA 100) in combination with a low-volume spray chamber (PFA Spray Chamber for Microflow, both from Elemental Scientific, Omaha, NE) was used to couple the µLC system to the ICPMS. The system was tuned by infusion of a 5 µM NaH2PO4/5 µM Na2SO4 aqueous solution. ESI-MS. Electrospray mass spectrometry was performed on a Q-Tof 2 instrument (Micromass, Manchester, U.K.), which was equipped with a nanoflow device. The spray voltage was ∼+1000 V. Spray capillaries for nanoESI were prepared in-house using a micropipet puller type P-87 (Sutter Instruments, Novato, CA) and coated with a semitransparent film of gold in a sputter unit. RESULTS AND DISCUSSION Strategy. Our new concept for accurate determination of the phosphorylation degree refers to phosphoproteins or phosphopeptides and is based on the simultaneous detection of sulfur and phosphorus using µLC-ICPMS. Although phosphopeptides do not generally contain cysteine or methionine, intact phosphoproteins show a high probability for the presence of at least one sulfur-containing residue. The sulfur signal serves as probe to quantify both the phospho and the dephospho form, whereas the phosphorus signal selectively quantifies the phospho form. To obtain a molar P/S ratio by this approach, three criteria have to be fulfilled: (i) interference-free detection of sulfur and phosphorus, (ii) knowledge of the influence of the LC solvent composition on detection sensitivity, and (iii) elution of the phosphoprotein as purified fraction from the µLC column. Interference-free detection of the main isotopes of sulfur (32S, natural abundance 95.012%) and phosphorus (31P, natural abundance 100%) requires a high-resolution mass analyzer operated at medium resolution. Products of ion-molecule reactions of ambient air with nominal masses of 31 and 32 are listed in Table 1. The data in Table 1 indicate that medium resolution is required for interference-free detection of 31P+ and 32S+ (see Experimental Section). When ICPMS is used as detector in a reversed-phase gradient LC procedure, the amount of organic modifier influences the ionization efficiency and the subsequent ion-molecule reactions, since the composition of the plasma is altered. For instance, it has been observed that the sulfur signal is suppressed with increasing amount of the organic solvent.20 To study these effects quantitatively, we have performed the following experiment: Solvents A and B used for gradient µLC-ICP were prepared containing 10 µM phosphate and 10 µM cysteine. Phosphate and (20) Jiang, S. J.; Houk, R. S. Spectrochim. Acta 1988, 438, 405-411.
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Figure 2. Determination of the phosphorylation degree of phosphoproteins by simultaneous measurement of P and S by µLCICPMS. (A) Calibration curve established using standard compounds (open squares) and determination of the stoichiometric P/S ratio in phosphoproteins (filled triangles); (B) visualization of the dephosphorylation experiment performed on R-casein by alkaline phosphatase (for details, see text and Table 2).
Figure 1. Determination of the stoichiometric P/S ratio from µLCICPMS data and measurement of the P/S ratio in an intact phosphoprotein. (A) Gradient µLC-ICPMS run with spiked mobile phases, containing 10 µM phosphate and 10 µM cysteine each, pure water was injected; (B) uncorrected P/S signal intensity ratio of the experiment displayed in (A), smoothed; (C) R-casein analysis by a gradient µLC-ICPMS run.
cysteine were selected as representative spikes for phosphorus and sulfur in a phosphoprotein for the following reasons: (i) the spikes contain phosphorus and sulfur in the analyte-equivalent oxidation status, and (ii) both compounds are highly hydrophilic avoiding an influence of the LC column on the P/S ratio. Using these spiked solvents, a standard gradient µLC run was performed and the signals for 31P and 32S were continuously monitored. Sample injection was replaced by injection of an equal volume of pure water. The result is displayed in Figure 1A. As expected, the ICP response for P and S is dependent on the solvent composition. Since this study is focused on accurate determination of the P/S ratio, we have calculated the experimental P/S ratio over the complete LC run time as displayed in Figure 1B. We have denominated this experimental P/S ratio as correction factor fcorr since normalization of an experimental P/S ratio by this fcorr factor results in the atomic P/S ratio. The corresponding calculation is given in formula 1. The plot in Figure 1B shows the
(PS)
1
exp fcorr
)
(PS)
atomic
(1)
dependence of the correction factor fcorr from the retention time. 3008 Analytical Chemistry, Vol. 73, No. 13, July 1, 2001
A P/S signal intensity ratio determined at a given retention time can be converted into an atomic P/S ratio using the appropriate fcorr value. In principle, oxygen and nitrogen could also be used for normalization of the phosphorus signal. However, these elements are characterized by poor sensitivity in ICPMS, and in addition, ambient air and solvents give rise to persistent background signals containing oxygen and nitrogen. Thus, sulfur and phosphorus represent the only elements in common amino acids that are targets for quantitative analysis by ICPMS. Phosphoproteins. To cover the relevant range of experimental P/S signal intensities, three gravimetric mixtures of phosphate and cysteine were prepared and infused at 4 µL/min into the ICPMS using the mobile-phase composition of the start of the LC gradient (5% B, fcorr ) 0.7). The resulting calibration plot is displayed in Figure 2A (open squares). Using this calibration plot, the degree of phosphorylation of various phosphoproteins was determined (Figure 2A, filled triangles). For the phosphoproteins analyzed by µLC-ICPMS, the R-casein analysis is displayed in Figure 1C as representative example. Among the phosphoproteins analyzed were R-casein (eight phosphorylated sites), β-casein (five phosphorylated sites) and three fractions of recombinant protein kinase A catalytic subunit containing two, three, and four phosphorylated sites.21 All these phosphoproteins analyzed by µLC-ICPMS elute in a narrow chromatographic retention time window at ∼35 min, so that the same fcorr value is valid for all phosphoproteins studied. Detailed numerical information on the calibration curve and the analysis of the intact phosphoproteins is summarized in Table 2. (21) Herberg, F. W.; Bell, S. M.; Taylor, S. S. Protein Eng. 1993, 6, 771-777.
Table 2. Theoretical Stoichiometric P/S Ratios of Investigated Phosphoproteins, Experimentally Observed P/S Ratios, Retention Time-Dependent Correction Factor, and Corrected P/S Ratios As Determined for Calibration Standards and Intact Phosphoproteinsa
sample blank mix 1 mix 2 mix 3 R-casein R-casein R-casein dephosphorylated β-casein β-casein PKA ×2P PKA ×3P PKA ×4P aThe
no. of 31P/ molecule
no. of 32S/ molecule
8 8
5 5 5
5 5 2 3 4
6 6 9 9 9
stioichiometric P/S ratio gravimetric*, theoretical** 0** 0.873* 1.5* 0.332* 1.6** 1.6** 0.83** 0.83** 0.22** 0.33** 0.44**
uncorrected P/S ratio
correction factor
0.65 1.10 0.25 2.96 2.91 2.5
0.7 0.7 0.7 1.7 1.7 1.7
1.58 1.66 0.42 0.62 0.86
1.7 1.7 1.7 1.7 1.7
corrected P/S ratio
ret time (min)
0 0.93 1.57 0.36 1.74 1.71 1.47 ∼85% left 0.93 0.98 0.25 0.40 0.50
0-9 0-9 0-9 35 35 35 35 35 35 35 35
data were extracted from the experimental data displayed in Figure 2.
Figure 3. Positive ion nanoESI mass spectra of R-casein. (A) Untreated sample consisting mainly of the 8-fold phosphorylated protein; (B) sample as in (A) but after partial dephosphorylation by alkaline phosphatase, showing conversion into a mixture of different phosphorylation states containing between three and eight phosphate groups. Satellite ions on the high-mass side represent sodium adducts.
The five above-mentioned phosphoproteins were also analyzed by nanoESI-MS, and the molecular weight observed was in agreement with the phosphorylation status expected from literature data. As representative example, Figure 3A shows the nanoESI spectrum of intact R-casein. The molecular ion containing eight phosphate groups plus corresponding sodium adducts represents the major ion signal.
In addition, minor signals of equal abundance corresponding to R-casein with seven or nine phosphate groups are also found. In summary, the molecular ion pattern of R-casein in Figure 3A justifies the assumption of 8-fold phosphorylation as the correct value. An aliquot of this sample of R-casein was subjected to partial dephosphorylation by phosphatase (see Experimental Section). The molecular ion pattern of the phosphatase-treated protein is given in Figure 3B. A mixture of differently phosphorylated R-casein species is observed, ranging from 3- to 8-fold phosphorylation. From the pattern of molecular ions including sodium adducts in Figure 3B, an average phosphate content of 5.48 phosphate/molecule () 70%) is found in phosphatase-treated R-casein compared to 8 phosphate/molecule () 100%) in untreated R-casein. An aliquot of the phosphatase-treated sample was analyzed by the standard µLC-ICPMS method introduced above. Based on the P/S atomic ratio as determined by this method, a phosphorylation degree of 85% compared to 100% of untreated R-casein was found. The effect is evident from Figure 3B, and exact numerical data are listed in Table 2. The deviation between the ESI-MS and the µLC-ICPMS data is in agreement with the phenomenon that in positive ion ESI-MS phosphorylation reduces the ionization efficiency. Phosphopeptides. To demonstrate the validity of the retention time-dependent correction factor, we analyzed compounds eluting at earlier retention times. For this purpose we selected two cysteine-containing synthetic phosphopeptides, TWpTLCGPEY and TWpTLCGTPEYLAPEIILSK. These peptides are derived from protein kinase A catalytic subunit and contain the autophosphorylation site Thr197. In the LC gradient used, these peptides elute at 29.5 and 34.0 min, respectively. These retention times correspond to fcorr values of 1.45 and 1.65, respectively. For the nonapeptide, a stoichiometric P/S ratio of 1:1 was determined. This finding was supported by ESI-MS, which confirmed the correct sequence and showed only minor impurities besides the abundant molecular ion signal. For the larger phosphopeptide with the sequence TWpTLCGTPEYLAPEIILSK, a stoichiometric P/S ratio of 0.75:1 was determined by µLC-ICPMS. Besides the correct molecular ion signals, the nanoESI spectrum of this Analytical Chemistry, Vol. 73, No. 13, July 1, 2001
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compound showed the presence of a deletion sequence in which the phosphothreonine residue was missing. This side product is frequently observed in the synthesis of peptides containing phosphothreonine. The identity of this side product was confirmed by sequencing using nanoESI-MS/MS. Calculation of the P/S ratio on the basis of the molecular ion signal intensities observed in the ESI mass spectrum resulted in a P/S ratio of 0.60:1. The deviation is caused by the less efficient formation of positive ions of ESI-MS for phosphopeptides compared to their unmodified analogues. To verify this effect, we performed dephosphorylation using barium hydroxide22 and redetermined the corresponding P/S ratio by measuring the ESI-MS peak intensity ratio of the H3PO4 elimination product and of the unaffected deletion sequence. This analysis resulted in a P/S ratio of 0.68.
CONCLUSIONS The results obtained analyzing the phosphoproteins and synthetic phosphopeptides confirm the compound-specific response of ESI-MS and show that µLC-ICPMS with simultaneous detection of phosphorus and sulfur is introduced as a new technology to deliver an accurate degree of phosphorylation for cysteine- and methionine-containing proteins and peptides. ACKNOWLEDGMENT We are indebted to H. P. Beck for encouraging support, to N. Ko¨nig and D. Bossemeyer for providing the sample of protein kinase A, and to R. Pipkorn for the synthetic phosphopeptides.
Received for review January 2, 2001. Accepted March 28, 2001. (22) Resing, K. A.; Johnson, R. S.; Walsh, K. A. Biochemistry 1995, 34, 94779487.
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AC010066S
