Mass Spectrometry Analysis of Phosphopeptides after Peptide

Sep 27, 2008 - Citation data is made available by participants in Crossref's Cited-by Linking service. For a more comprehensive list of citations to t...
0 downloads 0 Views 188KB Size
Download PDF
Anal. Chem. 2008, 80, 8324–8328

Mass Spectrometry Analysis of Phosphopeptides after Peptide Carboxy Group Derivatization Yawei Xu, Lijuan Zhang, Haojie Lu,* and Pengyuan Yang Department of Chemistry and Institutes of Biomedical Sciences, Fudan University, Shanghai 200433, P.R. China A nearly 100% yield peptide carboxy group derivatization method was offered to largely enhance phosphopeptide ionization efficiency. This method, adopting 1-(2-pyrimidyl) piperazine (PP) as the derivatization reagent, shows several advantages such as good reproducibility, ease of handling, rapid reaction time, and no side reaction. PP derivatization improves the hydrophobicities, pI values, and gas-phase basicities of peptides especially those of phosphopeptides. In the matrix assisted laser desorption ionization (MALDI) source, the ionization efficiencies of four synthetic phosphopeptides were increased by 50-101 times while that of three nonphosphopeptides were 10-40fold. In the electrospray ionization (ESI) source, PPderivatized phosphopeptides also gave much higher ionization efficiency improvements than nonphosphopeptides. When this method was applied to much more complex mixtures, tryptic BSA digests spiked with one single phosphopeptide in different molar ratios, the signal intensity of this phosphopeptide always had the largest increment among all those peptides. Obviously, this easily manipulated as well as highly specific method provides a promising tool for high-throughput phosphoproteome research. Phosphorylation is one of the most important post-translational modifications and plays a key role in biological processes such as cell degradation, signal transduction, and metabolic maintenance. As a reversible modification, only a trivial amount of proteins are transiently modified (1-2% of the entire amount of proteins present in phosphorylated form),1 the low substoichiometric nature of phosphopeptides, as well as their poor ionization efficiencies2,3 and the ion suppression effect by their unphosphorylated cognates, challenge the successful detection of phosphorylated peptides or proteins by mass spectrometry (MS). Several strategies have been introduced in the past decade to enrich phosphopeptides prior to MS detection. For example, Porath et al. developed immobilized metal ion (usually Fe3+, Ga3+) affinity chromatography (IMAC),4-6 Gygi and co-workers introduced * To whom correspondence should be addressed. E-mail: luhaojie@ fudan.edu.cn (1) Schlessinger, J. Harvey Lect. 1993, 89, 105–123. (2) Steen, H.; Kuster, B.; Fernandez, M.; Pandey, A.; Mann, M. Anal. Chem. 2001, 73, 1440–1448. (3) Jalili, P. R.; Sharma, D.; Ball, H. L. J. Am. Soc. Mass Spectrom. 2007, 18, 1007–1017. (4) Porath, J.; Carlsson, J.; Olsson, I.; Belfrage, G. Nature 1975, 258, 598– 599.

8324

Analytical Chemistry, Vol. 80, No. 21, November 1, 2008

strong cation exchange chromatography (SCX),7 and Weckwerth et al. applied metal oxide/hydroxide (usually Al(OH)3 and ZrO2) affinity chromatography (MOAC).8-10 Recently, Nakamura et al. adapted TiO2 columns as an alternative way for phosphopeptide enrichment.11 Those methods usually require esterification of acidic residues12 or adding certain acids (e.g., DHB) to the loading/washing buffer13 in order to obtain higher specificity. Other than the requirement of a large sample amount, these techniques have no effect on improving phosphopeptide ionization efficiency. As an alternative strategy, chemical derivatization was used to overcome the problems related to low ionization efficiencies of phosphopeptides. Unfortunately, most of these works published until now targeted exclusively phosphoserine and phosphothreonine,3,14-16 without covering phosphotyrosine which also plays a crucial role in many cell functions, for instance, receptor-mediated signaling pathways.17,18 Thus, there is a substantial need for a more rapid and general method for the analysis of protein phosphorylation. In the present study, we derivatize carboxy groups, which are always present in phosphopeptides independent of the amino acid residue, to develop an unambiguous, specific, and selective method in order to enhance the ionization efficiencies of phosphopeptides and reduce the suppression effects. According to previous studies, the main factors affecting the ionization efficiencies of peptides include both hydrophobicity and the pI value in the electrospray ionization (ESI) source.19 In the matrix assisted laser desorption ionization (MALDI) source, (5) Nuhse, T. S.; Stensballe, A.; Jensen, O. N.; Peck, S. C. Mol. Cell. Proteomics 2003, 2, 1234–1243. (6) Raska, C. S.; Parker, C. E.; Dominski, A.; Marzluff, W. F. Anal. Chem. 2002, 74, 3429–3433. (7) Ballif, B. A.; Villen, J.; Beausoleil, S. A.; Schwartz, D.; Gygi, S. P. Mol. Cell. Proteomics 2004, 3, 1093–1101. (8) Wolschin, F.; Wynkoop, S.; Weckwerth, W. Proteomics 2005, 5, 4389– 4397. (9) Kweon, H. K.; Hakansson, K. Anal. Chem. 2006, 8, 1743–1749. (10) Feng, S.; Yu, M. L.; Zhou, H. J. Mol. Cell. Proteomics 2007, 9, 1656–1665. (11) Sano, A.; Nakamura, H. Anal. Sci. 2004, 20, 861–864. (12) Ficarro, S. B.; McCleland, M. L.; Stukenberg, P. T.; Burke, D. J.; Ross, M. M.; Shabanowitz, J.; Hunt, D. F.; White, F. M. Nat. Biotechnol. 2002, 20, 301–305. (13) Larsen, M. R.; Thingholt, T. E.; Jensen, O. N.; Roepstorff, P.; Jørgensen, T. J. Mol. Cell. Proteomics 2005, 4, 873–886. (14) McLachlin, D. T.; Chait, B. T. Anal. Chem. 2003, 75, 6826–6836. (15) Arrigoni, G.; Resjo ¨, S.; Levander, F.; Nilsson, R.; Degerman, E.; Quadroni, M.; Pinna, L. A.; James, P. Proteomics 2006, 6, 757–766. (16) Tsumoto, H.; Ra, M.; Samejima, K.; Taguchi, R.; Kohda, K. Rapid Commun. Mass Spectrom. 2008, 7, 965–972. (17) Hunter, T. Philos. Trans. R. Soc. London, Ser. B 1998, 353, 583–605. (18) Hunter, T.; Sefton, B. Proc. Natl. Acad. Sci. U.S.A. 1980, 77, 1311–1315. (19) Pan, P.; Gunawardena, H. P.; Xia, Y.; McLuckey, S. A. Anal. Chem. 2004, 76, 1165–1174. 10.1021/ac801220c CCC: $40.75  2008 American Chemical Society Published on Web 09/27/2008

although the detailed mechanism has not been well understood, the gas-phase basicity is believed to contribute greatly to MALDI ionization efficiency: high gas-phase basicity results in considerable tendency of protonation of peptides.20 In comparison with nonphosphopeptides, phosphopeptides bear less hydrophobicities, lower pI values, or gas-phase basicities, which obviously results in lower ionization efficiencies. In this study, 1-(2-pyrimidyl) piperazine (PP) was chosen as the acidic residue derivatization reagent to improve peptide especially phosphopeptide ionization efficiencies in both ESI and MALDI sources, since the pyrimidyl groups could dramatically enhance the hydrophobicities, pI values, and gas-phase basicities. EXPERIMENTAL SECTION Materials and Chemicals. 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC, 95%), 1-hydroxy-7-azabenzotriazole (HOAt, 95%), and all synthetic peptides (95%) were obtained from GL Biochem (Shanghai, China). Bovine serum albumin (BSA, 95%), 2,5-dihydroxybenzoic acid (DHB, 98%), 1-(2pyrimidyl) piperazine (PP, 98%), and ammonium bicarbonate (ABC, 99.5%) were obtained from Sigma (St. Louis, MO). Acetonitrile (ACN, 99.9%) and trifluoroacetic acid (TFA, 99.8%) were purchased from Merck (Darmstadt, Germany). DMF was obtained from Shanghai No. 4 Reagent Company (Kunshan, China). Phosphoric acid (85%) was purchased from Shanghai Feida Chemical Reagents Ltd. (Shanghai, China). All these reagents were used as received without further purification. Deionized water (18.4 MΩ cm) used for all experiments was obtained from a Milli-Q system (Millipore, Bedford, MA). Synthesis of PP Derivatized Peptides. Solutions of PP (0.5%) in DMF (6 µL), EDC (2 mg/mL) in DMF (4 µL), HOAt (2 mg/ mL) in DMF (3 µL), and TFA (0.1%) in water (3 µL) were sequentially added to a solution of peptides or phosphopeptidespiked tryptic BSA (E100 ng/µL) in water (50 µL). The amount of TFA was especially critical to this reaction and needed to be carefully adjusted to keep the final pH between 7.5 and 7.8. After vortexing for several seconds at room temperature, the solvents were removed by vacuum centrifuge to terminate the PPderivatization reaction. The modification of PP-derivatization was stable in water or TFA solution (E2%) for at least 48 h (Supporting Information Figures 1-3). Extra EDC and HOAt were not able to react with carboxy groups in the solution of water or 50% CH3CN/0.1% TFA. PP-derivatized peptides can then be directly applied for MALDI-MS or ESI-MS analysis without any additional cleanup step. MALDI Mass Spectrometry. The MALDI mass spectra were acquired with an Applied Biosystems 4700 proteomics analyzer instrument. The matrix solution (10 mg/mL CHCA in 50% CH3CN/0.1% TFA, 0.5 µL) was deposited to and dried on the MALDI probe, and the peptide solution (0.5 µL in 50% CH3CN/ 0.1% TFA) was then deposited. ESI Mass Spectrometry. LC-MS/MS of the peptide mixture was performed on a Bruker Daltonics (Billerica, MA) HCTultra PTM Discovery quadrupole ion trap mass spectrometer. The ion trap mass spectrometer was coupled with a Dionex Ultimate 3000 nano-LC system (Sunnyvale, CA) through a nanoelectrospray interface (Bruker Daltonics). The nano-LC was equipped with a (20) Dreisewerd, K. Chem. Rev. 2003, 103, 395–425.

Scheme 1. Synthesis of 1-(2-Pyrimidyl)piperazine Derivatized Peptidea

a EDC ) 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, HOAt ) 1-hydroxy-7-azabenzotriazole, TFA ) trifluoroacetic acid.

set of Dionex PepMap C18 precolumn (300 µm × 5 mm; particle size 50 µm) and a Dionex PepMap C18 column (75 µm × 150 mm; particle size 3 µm). The peptide mixture was dissolved in 0.1% FA solution. For each LC-MS/MS run, typically 6 µL of sample solution was loaded to the precolumn and washed with loading buffer (0.1% FA-H2O) for 3 min before injecting into the LC column. The buffers used for LC separation are buffer A (0.1% FA-H2O) and buffer B (0.1% FA-ACN).The LC gradient was conducted as follows: 3% B to 35% B in 20 min, 35% B to 95% B in 4 min, and stayed at 95% B for 8 min. The column was finally re-equilibrated with 3% B for 15 min before the next run. The flow was set up at 350 nL/min. RESULTS AND DISCUSSION A major requirement for an attractive derivatization method is high conversion ratio of modification reaction. Following previous literature,21 we chose peptide coupling reagents (EDC and HOAt) to convert reactants as much as possible in the DMF solution. However, yield as close as possible to 100% had never been reached when following the published papers. Once the normally used DMF was replaced with water and a certain amount of TFA was added to adjust the pH value of the reaction system, all carboxy groups of peptides, including the C-terminal aspartic and glutamic acids, can be almost fully converted to their PP derivatized cognates (Scheme 1, Figure 1). (More quantitative data of reaction yield could be found in Table 1 in the Supporting Information.) Our data indicates that the optimized pH value for a complete reaction is 7.5-7.8. Moreover, the reaction time was shortened to a few seconds. Vortexing by hand under room temperature was enough for this reaction to complete. This adjustment rendered the reaction prominent features to make it a potentially powerful tool: ease of handling, rapid reaction time, good reproducibility, no side reaction, and free of harsh treatment like high temperature or sonication. The gentle reaction conditions keep peptides away from any potential threat of degradation. Furthermore, no side reaction product was found throughout the whole process, suggesting the high reaction specificity between PP and the carboxy group (Figure 1). Vacuum centrifuge was adopted to terminate the reaction by removing the solvents. The stability of the modification is rather good in water or TFA solution (E2%) for at least 48 h (Supporting Information Figures 1-3). Extra reagents would not react with peptides nor be detected by MALDI-MS or ESI-MS, so no additional cleanup step is needed before MS analysis. (21) Ho, G. J.; Emerson, K. M.; Mathre, D. J. J. Org. Chem. 1995, 60, 3569– 3570.

Analytical Chemistry, Vol. 80, No. 21, November 1, 2008

8325

Figure 1. MALDI mass spectra of synthetic peptides (a) KRGSGAW and (b) DRVYIHPF after derivatization. X-PP represents the PP-derivatized amino acid.

Figure 2. MALDI mass spectra of equimolar mixtures of native and derivatized nonphosphopeptide (a) and phosphopeptide (b) with CHCA matrix. X-PP represents the PP-derivatized amino acid.

As a first step, we used MALDI-MS to evaluate the improvements of ionization efficiency because peptides ionized in the ESI source often present more than one charge state, which complicates the evaluation process. We first derivatized the model peptide bradykinin (RPPGFSP) following the approach described in the experimental section, and mixed the derivatized bradykinin with its equimolar underivatized counterpart for subsequent MALDI-MS analysis (matrix: R-cyano-4-hydroxycinnamic acid (CHCA)). The intensity of the peak at m/z 903.4 corresponding to the PP-derivatized bradykinin was 12.1 times higher than that of its underivatized cognate (m/z 757.3) (Figure 2a). Obviously, the PP-derivatized peptide has much higher absolute intensity and 8326

Analytical Chemistry, Vol. 80, No. 21, November 1, 2008

ionization efficiency than its underivatized cognate. To test this effect on phosphopeptides, we synthesized a phosphopeptide (TPpTAPSLG) and treated it the same way as bradykinin. The intensity of this phosphopeptide was increased by 101.2 times through PP derivatization (Figure 2b). To further examine the effectiveness of our method, we synthesized another two nonphosphopeptides (KRGSGAW and NRGSGAW) and three phosphopeptides (DPpTAPSLG, TPpTAPpSLG, and DPpTAPpSLG). Each peptide was treated exactly the same way as the two peptides mentioned above. With all seven peptides, we found the ionization efficiencies of phosphopeptides were increased by 50 to over 100 times (Figure 3b) while that of nonphosphopeptides were only

Figure 3. Y axis ) (S/N of PP-derivatized peptide)/(S/N of its native cognate). Error bar represents the standard deviation of six distinct results. Three nonphosphopeptides (a) and four phosphopeptides (b) were tested.

Figure 5. MALDI mass spectra of mixtures of a single phosphopeptide and tryptic BSA digests with CHCA matrix before (a) and after (b-d) PP derivatization. The same Arabic numerals in both parts a and b-d indicate the same peptide of BSA. Asterisks represent newly detected peptides from BSA after derivatization. X-PP represents the PP-derivatized amino acid.

Figure 4. Extracted ion chromatograms of the native peptide and its PP-derivatized cognate. X-PP represents the PP-derivatized amino acid.

10-40-fold (Figure 3a). We then examined the ionization effects of PP-derivatized peptides on the ESI source. A peptide mixture consisting of four peptides (two nonphosphopeptides and two phosphopeptides) and their equimolar PP-derivatized counterparts was subjected to LC-ESI-MS analysis. All PP-derivatized peptides show longer retention time (Figure 4) since the hydrophobicity and the pI value were all increased simultaneously through PPderivatization. The extracted ion chromatograms in Figure 4 also clearly demonstrated that PP-derivatized peptides have higher ionization efficiencies. Furthermore, the increments of ionization

efficiency for phosphopeptides (Figure 4a,b) were obviously larger than those of nonphosphopeptides (Figure 4c,d). Accordingly, in both ESI and MALDI mass spectra, phosphopeptides revealed much larger increments on ionization efficiencies than nonphosphopeptides after PP derivatization. A conclusion could thus be made that PP-derivatization can not only largely enhance the ionization efficiencies of peptides especially phopsphopeptides (no matter if one or two phosphorylation site(s) and derivatization site(s)) but also overcome the problem of the suppression effect on phosphopeptides by their unmodified cognates. To examine the feasibility of our method on a much more complex system, we mixed a phosphopeptide (TPpTAPSLG) with tryptic BSA digests in a molar ratio of 133:1 (2 pmol/µL: 15 fmol/ µL). Although the amount of the phosphopeptide was 132 times more than that of BSA digests, it was almost undetectable in the MALDI-MS spectrum (Figure 5a). This clearly indicated the relatively low ionization efficiency of phosphopeptide and the suppression effect by nonphosphopeptides. However, after PPderivatization, the signals of most peptides were intensified and the phosphopeptide gave the highest signal intensity (Figure 5b). When the molar ratio of phosphopeptide (TPpTAPSLG) and BSA digests decreased to 6:1, the signal intensity of the phosphopeptide Analytical Chemistry, Vol. 80, No. 21, November 1, 2008

8327

was still higher than most of the nonphosphopeptides (Figure 5c). Furthermore, even tryptic BSA digests were equimolar mixed with the phosphopeptide, the signal intensity of this phosphopeptide was still comparable with many other nonphosphopeptides after PP-derivatization (Figure 5d). It clearly shows that PP-derivatization is very effective in a complex system such as tryptic digest proteins since both the problems of low ionization efficiency and suppression effect could be overcome. In summary, we successfully modified carboxy groups of peptides with 1-(2-pyrimidyl)piperazine (PP) to enhance the ionization efficiencies of peptides especially that of phosphopeptides and make them MS detectable without any specific enrichment. Once carboxy groups were derivatized with PP, the hydrophobicities, pI values, and gas-phase basicities of phosphopeptides were largely increased so that the ionization efficiencies could be dramatically enhanced accordingly. As PP derivatization neutralizes the extra negative charges and decreases the hydrophilicities brought in by phosphate groups, the ionization efficiencies of phosphopeptides can be increased up to 101-fold. Comparing with other methods, this improved method needs less sample amount, costs less time, and lacks any drawback of traditional derivatization methods such as low yields, harsh

8328

Analytical Chemistry, Vol. 80, No. 21, November 1, 2008

reaction conditions, and unknown side reactions. This new method has been applied to both standard peptides and tryptic protein digests and was proved to be very efficient and extremely powerful. We also expect it will be very promising in large-scale phosphoproteome and proteome analysis in the near future. ACKNOWLEDGMENT This work was supported by the National Science and Technology Key Project of China (Grants 2007CB914100, 2009CB825607, 2008ZX10207, 2006AA02Z134, and 2006AA02A308), National Natural Science Foundation of China (Grants 20735005, 20875016, 30672394, and 30530040), NCET, Shanghai Leading Academic Discipline (Grant B109), and Shanghai Rising-Star Program (Grant 06QA14004). SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review June 16, 2008. Accepted August 25, 2008. AC801220C