Differentiation of Phosphorylated and Unphosphorylated Peptides by

Anal. Chem. 2005, 77, 5726-5734

Differentiation of Phosphorylated and Unphosphorylated Peptides by High-Performance Liquid Chromatography-Electrospray Ionization-Infrared Multiphoton Dissociation in a Quadrupole Ion Trap Matthew C. Crowe and Jennifer S. Brodbelt*

Department of Chemistry and Biochemistry, The University of Texas at Austin, Austin, Texas 78712

Infrared multiphoton dissociation (IRMPD) in a quadrupole ion trap coupled to high-performance liquid chromatography allows the selective dissociation of phosphorylated peptides in mixtures following chromatographic separation. This method is shown to be effective for differentiation of phosphorylated peptides from unphosphorylated ones; only the abundances of the phosphorylated species are appreciably decreased following exposure to 125 ms of 10.6-µm radiation. This LC-IRMPDMS strategy is demonstrated for a mock mixture of peptides and a tryptic digest of rS1-casein. The ability of this technique to differentiate peptides based on phosphorylation state is unaffected by whether the peptides are protonated or sodium-cationized. One of the most important and difficult aspects of proteomics is the determination of when, where, and to what extent posttranslation modifications take place. Of the many types of posttranslational modifications (sulfonation, phosphorylation, glycosylation, acetylation, methylation, formation of disulfide linkages, etc.), phosphorylation, which may occur on serine, threonine, and tyrosine residues, is one of the most important.1,2 This reversible covalent modification is involved in the regulation of cellular processes such as metabolism, growth, reproduction, and differentiation.3-5 Because of the significance of phosphorylation, it is important to develop highly sensitive analytical techniques capable of identifying the presence, absence, and location of this modification, particularly for complex mixtures. The speed and sensitivity of mass spectrometry, particularly tandem mass spectrometry, make it a versatile method for determining the primary sequence of a protein of interest as well as for the identification and location of posttranslational modifications.6-30 Various tandem mass spectrometry techniques have been used to identify phosphorylation sites on peptides and * Corresponding author. Phone: 512-471-0028. E-mail: jbrodbelt@ mail.utexas.edu. (1) Faux, M. C.; Scott, J. D. Trends Biochem. Sci. 1996, 21, 312-315. (2) Sun, H.; Tonks, N. K. Trends Biochem. Sci. 1994, 19, 480-485. (3) Krebs, E. G. Trends Biochem. Sci. 1994, 19, 439-439. (4) Hunter, T. Cell 1995, 80, 225-236. (5) Posada, J.; Cooper, J. A. Mol. Biol. Cell 1992, 3, 583-592. (6) Biemann, K. Biomed. Environ. Mass Spectrom. 1988, 16, 99-111.

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proteins. Collisionally activated dissociation (CAD) can be used to reveal a site of phosphorylation based on unique mass losses associated with phosphorylated peptides.9,14,19,23 In MALDI-TOF mass spectrometry, similar fragments can be observed upon postsource decay (PSD) of metastable ions formed in the source region.17 Recently, several techniques using methods other than the neutral loss of phosphate to locate a site of phosphorylation have been developed, such as electron-capture dissociation (7) Carr, S. A.; Hemling, M. E.; Bean, M. F.; Roberts, G. D. Anal. Chem. 1991, 63, 2802-2824. (8) Cohen, P.; Gibson, B. W.; Holmes, C. F. B. Methods Enzymol. 1991, 201, 153-168. (9) Huddleston, M. J.; Annan, R. S.; Bean, M. F.; Carr, S. A. J. Am. Soc. Mass Spectrom. 1993, 4, 710-717. (10) Affolter, M.; Watts, J. D.; Krebs, D. L.; Aebersold, R. Anal. Biochem. 1994, 223, 74-81. (11) Burlingame, A. L.; Boyd, R. K.; Gaskell, S. J. Anal. Chem. 1994, 66, R634R683. (12) Hunter, A. P.; Games, D. E. Rapid Commun. Mass Spectrom. 1994, 8, 559570. (13) Resing, K. A.; Johnson, R. S.; Walsh, K. A. Biochemistry 1995, 34, 94779487. (14) Carr, S. A.; Huddleston, M. J.; Annan, R. S. Anal. Biochem. 1996, 239, 180192. (15) Busman, M.; Schey, K. L.; Oatis, J. E.; Knapp, D. R. J. Am. Soc. Mass Spectrom. 1996, 7, 243-249. (16) Ladner, R. D.; Carr, S. A.; Huddleston, M. J.; McNulty, D. E.; Caradonna, S. J. J. Biol. Chem. 1996, 271, 7752-7757. (17) Annan, R. S.; Carr, S. A. Anal. Chem. 1996, 68, 3413-3421. (18) Qin, J.; Chait, B. T. Anal. Chem. 1997, 69, 4002-4009. (19) DeGnore, J. P.; Qin, J. J. Am. Soc. Mass Spectrom. 1998, 9, 1175-1188. (20) Neubauer, G.; Mann, M. Anal. Chem. 1999, 71, 235-242. (21) Stensballe, A.; Jensen, O. N.; Olsen, J. V.; Haselmann, K. F.; Zubarev, R. A. Rapid Commun. Mass Spectrom. 2000, 14, 1793-1800. (22) Flora, J. W.; Muddiman, D. C. Anal. Chem. 2001, 73, 3305-3311. (23) Schlosser, A.; Pipkorn, R.; Bossemeyer, D.; Lehmann, W. D. Anal. Chem. 2001, 73, 170-176. (24) Shi, S. D. H.; Hemling, M. E.; Carr, S. A.; Horn, D. M.; Lindh, I.; McLafferty, F. W. Anal. Chem. 2001, 73, 19-22. (25) Flora, J. W.; Muddiman, D. C. J. Am. Chem. Soc. 2002, 124, 6546-6547. (26) Wind, M.; Kelm, O.; Nigg, E. A.; Lehmann, W. D. Proteomics 2002, 2, 15161523. (27) Chalmers, M. J.; Quinn, J. P.; Blakney, G. T.; Emmett, M. R.; Mischak, H.; Gaskell, S. J.; Marshall, A. G. J. Proteome Res. 2003, 2, 373-382. (28) Flora, J. W.; Muddiman, D. C. J. Am. Soc. Mass Spectrom. 2004, 15, 121127. (29) Chalmers, M. J.; Kolch, W.; Emmett, M. R.; Marshall, A. G.; Mischak, H. J. Chromatogr., B 2004, 803, 111-120. (30) Chalmers, M. J.; Hakansson, K.; Johnson, R.; Smith, R.; Shen, J. W.; Emmett, M. R.; Marshall, A. G. Proteomics 2004, 4, 970-981. 10.1021/ac0509410 CCC: $30.25

© 2005 American Chemical Society Published on Web 08/02/2005

(ECD),31-37 which results in backbone fragmentation without the loss of the phosphate group.21,24,29-31 The fragments produced by ECD are typically of low relative abundance but provide extensive sequence information (c and z ions) complementary to that obtained with thermal dissociation techniques such as CAD and infrared multiphoton dissociation (IRMPD) (b and y ions). To circumvent the difficulties in implementing ECD on a quadrupole ion trap, Syka and co-workers have developed electron-transfer dissociation (ETD), which allows the production of ECD-like fragment ions in a linear quadrupole ion trap.38 This technique has been utilized for phosphopeptide analysis and has been shown to produce predominantly backbone cleavage product ions, allowing identification of peptide phosphorylation sites.38 The drawbacks to this technique are that it requires relatively complex scan functions (necessary for the simultaneous trapping of ions of both polarities and reaction thereof), multiple ionization sources, and results, like ECD, in fragment ions of relatively low abundance when compared to those obtained by IRMPD and CAD. Because of the unique absorption properties of 10.6-µm light characteristic of the phosphate group,39 IRMPD be used to circumvent some of the problems encountered with other activation techniques. Methods that rely on common neutral losses for the detection of phosphopeptides are less reliable for the detection of tyrosine phosphorylated peptides than for serine or threonine phosphorylated peptides because tyrosine residues lose the phosphate group less readily than do serine and threonine.10,22 In addition, methods relying on PSD, ECD, or ETD generate fragment ions at relatively low abundance while typical IRMPD dissociation efficiency can approach 100%. Infrared multiphoton dissociation has been utilized with much success in FTICR mass spectrometry.22,25,27,28,35,40-48 Building on the initial small molecule FTICR-IRMPD studies by Beauchamp and co-workers49 and Eyler (31) Zubarev, R. A. Curr. Opin. Biotechnol. 2004, 15, 12-16. (32) Zubarev, R. A.; Kelleher, N. L.; McLafferty, F. W. J. Am. Chem. Soc. 1998, 120, 3265-3266. (33) Kelleher, R. L.; Zubarev, R. A.; Bush, K.; Furie, B.; Furie, B. C.; McLafferty, F. W.; Walsh, C. T. Anal. Chem. 1999, 71, 4250-4253. (34) Horn, D. M.; Ge, Y.; McLafferty, F. W. Anal. Chem. 2000, 72, 4778-4784. (35) Hakansson, K.; Cooper, H. J.; Emmett, M. R.; Costello, C. E.; Marshall, A. G.; Nilsson, C. L. Anal. Chem. 2001, 73, 4530-4536. (36) Hakansson, K.; Emmett, M. R.; Hendrickson, C. L.; Marshall, A. G. Anal. Chem. 2001, 73, 3605-3610. (37) McLafferty, F. W.; Horn, D. M.; Breuker, K.; Ge, Y.; Lewis, M. A.; Cerda, B.; Zubarev, R. A.; Carpenter, B. K. J. Am. Soc. Mass Spectrom. 2001, 12, 245-249. (38) Syka, J. E. P.; Coon, J. J.; Schroeder, M. J.; Shabanowitz, J.; Hunt, D. F. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 9528-9533. (39) Silverstein, R. M.; Bassler, G. C.; Morrill, T. C. Spectrometric Identification of Organic Compounds; Wiley: New York, 1981; p 170. (40) Little, D. P.; Aaserud, D. J.; Valaskovic, G. A.; McLafferty, F. W. J. Am. Chem. Soc. 1996, 118, 9352-9359. (41) Little, D. P.; Speir, J. P.; Senko, M. W.; O’Connor, P. B.; McLafferty, F. W. Anal. Chem. 1994, 66, 2809-2815. (42) Hofstadler, S. A.; Sannes-Lowery, K. A.; Griffey, R. H. Anal. Chem. 1999, 71, 2067-2070. (43) Shi, S. D. H.; Hendrickson, C. L.; Marshall, A. G.; Siegel, M. M.; Kong, F. M.; Carter, G. T. J. Am. Soc. Mass Spectrom. 1999, 10, 1285-1290. (44) Li, W. Q.; Hendrickson, C. L.; Emmett, M. R.; Marshall, A. G. Anal. Chem. 1999, 71, 4397-4402. (45) Hakansson, K.; Hudgins, R. R.; Marshall, A. G. J. Am. Soc. Mass Spectrom. 2003, 14, 23-41. (46) Drader, J. J.; Hannis, J. C.; Hofstadler, S. A. Anal. Chem. 2003, 75, 36693674. (47) Hofstadler, S. A.; Drader, J. J.; Gaus, H.; Hannis, J. C.; Sannes-Lowery, K. A. J. Am. Soc. Mass Spectrom. y 2003, 14, 1413-1423. (48) Xie, Y. M.; Lebrilla, C. B. Anal. Chem. 2003, 75, 1590-1598.

and co-workers,50 McLafferty and co-workers have shown it possible to effectively dissociate and sequence proteins41 and oligonucleotides40 in an FTICR analyzer. IRMPD in an FTICR has also been applied for the fragmentation of the glycan components of glycopeptides,35 to validate the structures of saccharomicins,43 and for the tandem mass spectral analysis of alkali metalcoordinated oligosaccharides.48 Because of the suitably low background pressures used in FTICR mass spectrometry (which minimizes energy loss due to collisions with background gas neutrals), IRMPD is better suited for use in these types of mass analyzers than are collisional techniques, which require extra gas loads for optimal results41 and an accompanying period for the removal of the collision gas.48 The other trapping mass spectrometer in which IRMPD has been explored is the quadrupole ion trap.51-63 The quadrupole ion trap is typically operated with ∼1 mTorr helium background gas for optimum instrument performance;64 the presence of this background gas makes the QIT particularly well-suited for collisional activation. However, because of deactivating collisions that take place at these pressures, this environment is less wellsuited for IRMPD than low-pressure FTICR systems. IRMPD in a quadrupole ion trap has been pursued despite this fundamental drawback because it has several important advantages over the more conventional collisional activation technique. First, the low m/z cutoff (approximately one-third of the m/z value of the precursor ion) necessary for efficient CAD is not required in IRMPD experiments. This loss of the lower one-third of the m/z range in CAD experiments is related to the increase in the level of the rf voltage needed to trap the selected precursor ions efficiently during collisional activation; unfortunately, this increase in precursor ion stability occurs at the expense of storage of the lower m/z fragment ions. Thus, IRMPD expands the effective m/z range of the ion trap instrument for the analysis of fragment ions.53 Second, for multiple stages of mass spectrometry (MSn), IRMPD is more efficient than CAD since IRMPD requires no alteration of the stable trajectory or kinetic energy of the trapped ions for excitation, as is necessary for CAD. This reduces the extent of ion loss through scattering relative to collisional activation. Last, (49) Bomse, D. S.; Berman, D. W.; Beauchamp, J. L. J. Am. Chem. Soc. 1981, 103, 3967-3971. (50) Baykut, G.; Watson, C. H.; Weller, R. R.; Eyler, J. R. J. Am. Chem. Soc. 1985, 107, 8036-8042. (51) Stephenson, J. L.; Booth, M. M.; Shalosky, J. A.; Eyler, J. R.; Yost, R. A. J. Am. Soc. Mass Spectrom. 1994, 5, 886-893. (52) Stephenson, J. L.; Booth, M. M.; Boue, S. M.; Eyler, J. R.; Yost, R. A. Biochem. Biotechnol. Appl. Electrospray Ionization Mass Spectrom. 1996, 619, 512564. (53) Colorado, A.; Shen, J. X. X.; Vartanian, V. H.; Brodbelt, J. Anal. Chem. 1996, 68, 4033-4043. (54) Goolsby, B. J.; Brodbelt, J. S. J. Mass Spectrom. 1998, 33, 705-712. (55) Goolsby, B. J.; Brodbelt, J. S. J. Mass Spectrom. 2000, 35, 1011-1024. (56) Shen, J.; Brodbelt, J. S. Analyst 2000, 125, 641-650. (57) Payne, A. H.; Glish, G. L. Anal. Chem. 2001, 73, 3542-3548. (58) Gabryelski, W.; Li, L. Rapid Commun. Mass Spectrom. 2002, 16, 18051811. (59) Crowe, M. C.; Brodbelt, J. S.; Goolsby, B. J.; Hergenrother, P. J. Am. Soc. Mass Spectrom. 2002, 13, 630-649. (60) Hashimoto, Y.; Hasegawa, H.; Yoshinari, K.; Waki, I. Anal. Chem. 2003, 75, 420-425. (61) Keller, K. M.; Brodbelt, J. S. Anal. Chem. 2004, 326, 200-210. (62) Goolsby, B. J.; Brodbelt, J. S. Anal. Chem. 2001, 73, 1270-1276. (63) Vartanian, V. H.; Goolsby, B.; Brodbelt, J. S. J. Am. Soc. Mass Spectrom. 1998, 9, 1089-1098. (64) Stafford, G. C.; Kelley, P. E.; Syka, J. E. P.; Reynolds, W. E.; Todd, J. F. J. Int. J. Mass Spectrom. Ion Processes 1984, 60, 85-98.

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the nonresonant IRMPD technique results in formation of secondary and higher order fragments, which can provide additional structural information beyond that obtained in a single resonant collisional activation experiment. Flora and Muddiman have shown that it is possible to selectively dissociate phosphorylated peptides with IRMPD in an FTICR mass analyzer.22,25,28 In a previous publication, we reported that it is possible to perform these types of experiments, including tryptic digests of phosphoproteins, in a quadrupole ion trap.65 Our work and that of Flora and Muddiman showed that the selective dissociation of phosphorylated peptides in complex mixtures using 10.6-µm light is widely applicable. To reduce the spectral complexity and at the same time alleviate ESI signal suppression of polar analytes, liquid chromatography has been coupled to mass spectrometry for the identification of phosphorylated peptides in a variety of studies. Vissers and co-workers have applied tandem mass spectrometry in a quadrupole orthogonal acceleration time-of-flight (Q-TOF) mass spectrometer following liquid chromatographic separations for the identification of phosphorylated peptides via neutral losses and the formation of characteristic immonium ions.66 Using collisionally activated dissociation in a quadrupole ion trap, Lee and coworkers have applied LC/MS/MS for the identification of phosphorylated species in activating transcription factor-2.67 To combat the minimal amount of phosphorylation found in proteins, Hunt’s group has published work involving the selective enrichment of phosphopeptides prior to LC/MS/MS analysis, improving sensitivity for these species.68 Mann and coauthors have also outlined methods of selective phosphopeptide enrichment as well as LC and MS phosphoproteomics methodologies in a recent review.69 Work by Tanaka and co-workers has shown nano-LC-IRMPD in an FTICR analyzer is useful for the identification of proteins following proteolytic digestion.70 Previous work by Marshall and co-workers has shown that the combination of liquid chromatography and IRMPD in an FTICR analyzer is useful for the rapid identification of intact proteins in mixtures through tandem mass spectrometry and database searching.44 They have successfully applied this method for the identification of four previously unidentified phosphorylation sites in protein kinase C.27 In addition, Kelleher and co-workers have successfully demonstrated that IRMPD in an FTICR instrument following reversed-phase separations is useful for the identification of intact proteins following two-dimensional gel fractionation.71-75 (65) Crowe, M. C.; Brodbelt, J. S. J. Am. Soc. Mass Spectrom. 2004, 15, 15811592. (66) Bateman, R. H.; Carruthers, R.; Hoyes, J. B.; Jones, C.; Langridge, J. I.; Millar, A.; Vissers, J. P. C. J. Am. Soc. Mass Spectrom. 2002, 13, 792-803. (67) Tsay, Y. G.; Wang, Y. H.; Chiu, C. M.; Shen, B. J.; Lee, S. C. Anal. Biochem. 2000, 287, 55-64. (68) Garcia, B. A.; Shabanowitz, J.; Hunt, D. F. Methods 2005, 35, 256-264. (69) Mann, M.; Ong, S. E.; Gronborg, M.; Steen, H.; Jensen, O. N.; Pandey, A. Trends Biotechnol. 2002, 20, 261-268. (70) Kosaka, T.; Yoneyama-Takazawa, T.; Kubota, K.; Matsuoka, T.; Sato, I.; Sakaki, T.; Tanaka, Y. J. Mass Spectrom. 2003, 38, 1281-1287. (71) Meng, F. Y.; Cargile, B. J.; Patrie, S. M.; Johnson, J. R.; McLoughlin, S. M.; Kelleher, N. L. Anal. Chem. 2002, 74, 2923-2929. (72) Forbes, A. J.; Patrie, S. M.; Taylor, G. K.; Kim, Y. B.; Jiang, L. H.; Kelleher, N. L. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 2678-2683. (73) Du, Y.; Meng, F. Y.; Patrie, S. M.; Miller, L. M.; Kelleher, N. L. J. Proteome Res. 2004, 3, 801-806. (74) Patrie, S. M.; Robinson, D. E.; Meng, F. Y.; Du, Y.; Kelleher, N. L. Int. J. Mass Spectrom. 2004, 234, 175-184.

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The work presented here illustrates that IRMPD tandem mass spectrometry in conjunction with high-performance liquid chromatography (HPLC)-MS in a quadrupole ion trap allows the selective dissociation of phosphorylated peptides in complex mixtures, particularly tryptic digests of phosphoproteins. The LCESI-IRMPD-MS technique allows for rapid, reliable screening of enzymatic digests of phosphorylated proteins. EXPERIMENTAL SECTION Mass Spectrometry. All mass spectrometry experiments were performed on a modified M-8000 Hitachi 3DQ ion trap mass spectrometer (Hitachi Ltd.) using the 3DQ software package and the standard ESI source. Electrospray ionization and ion trapping conditions were optimized using a three-component peptide mixture and were not changed from sample to sample. The pressure in the analyzer region was nominally 5.0 × 10-5 Torr, which has been found to be optimal for instrument sensitivity and resolution. Solutions of 10 µM analyte in 50:50 H2O/MeOH were loaded onto the analytical column with a Hitachi autosampler (model L-7200); injection volumes ranged from 5 to 10 µL. Infrared Multiphoton Dissociation. IRMPD experiments were performed with a Synrad 50-W continuous wave CO2 laser (series 48, model 48-5; Synrad, Mukilteo, WA). The laser output gating was controlled by TTL triggers set up in the software. The trapping volume of the quadrupole ion trap was irradiated through a ZnSe window mounted on the vacuum chamber aligned with a 4-mm hole in the ring electrode. For selective LC-IRMPD-MS experiments, the irradiation time (t ) 125 ms) and power (50 W) were kept constant in order to allow differentiation of phosphorylated and unphosphorylated species. The laser power utilized in these experiments is high relative to that used in FTICR analyzers due to the high background pressure necessary for optimum trapping performance and the deactivating collisions associated with these higher pressures. In addition, the laser output was not focused (diameter, 4.0 mm) in order to easily encompass the trapped ion packet (diameter, ∼1.0 mm), which also increases the power necessary for successful IRMPD experiments. Analytical High-Performance Liquid Chromatography. Liquid chromatography was performed on a Hitachi L-7000 system, including an L-7100 pump, L-7200 UV detector, and an L-7000 autosampler, all of which were controlled by the Hitachi 3DQ software. Reversed-phase analytical separations took place on a Polaris (Varian Inc., Torrance, CA) C18-A column (50 × 2.0 mm, 3-µm packing) equipped with a Polaris C18-A MetaGuard column (10 × 2.0 mm, 3-µm packing). For analytical separations, a flow rate of 200 µL/min was used with gradient elutions using solvents A (H2O, 0.1% formic acid) and B (MeOH, 0.1% formic acid). Analyte detection was accomplished with the M-8000 mass analyzer as well as the L-7200 UV detector (λ ) 280 nm). Preparative HPLC. Purified RS1-casein was obtained by preparative HPLC of an 85% pure R-casein sample purchased from Sigma-Aldrich (St. Louis, MO). The preparative HPLC method was performed on an A¨ KTA purifier (Amersham Biosciences, Piscataway, NJ) using a self-packed perfusion column Poros 10 R2 RP C18 4.6 × 100 mm (Applied Biosystems, Foster City, CA) (75) Meng, F. Y.; Du, Y.; Miller, L. M.; Patrie, S. M.; Robinson, D. E.; Kelleher, N. L. Anal. Chem. 2004, 76, 2852-2858.

using 150-µL injections of a 2 mg/mL solution of R-casein dissolved in 18 MΩ H2O. The mobile-phase gradient used was 10% B for 6 min, 10-39% B over 1.5 min, 39-49% B over 36.5 min, 49-100% for 1.5 min, and 100% B for 2.5 min at a flow rate of 1.0 mL/min. (A: 0.1% trifluoroacetic acid in 18 MΩ H2O. B: 0.02% TFA in 20% H2O, 80% acetonitrile). Effluent monitoring was performed using UV detection at 280 nm. Polyacrylamide Gel Electrophoresis. Polyacrylamide gel electrophoresis (PAGE) was performed on a NuPAGE 4-12% BisTris Gradient Gel (Novex, San Diego, CA). The run buffer utilized was 50 mM MES-Tris, 3.465 mM SDS, 1.025 mM EDTA. PAGE experiments were performed on 10-50 µg of protein sample dissolved in the run buffer at pH 7.3. A 200-V potential difference was applied to the gel for ∼1 h/analysis. Gel staining was performed for 15-30 min in 50% methanol, 10% acetic acid + coomassie blue R250 (1 g/L). Destaining was performed for >10 min in 50% methanol, 10% acetic acid. The gel images were obtained with a standard color scanner. Enzymatic Digestion. All proteins were enzymatically digested with immobilized trypsin in 50 mM ammonium bicarbonate buffer (pH 8.0) for 14-18 h. at 37 °C. Following each reaction, the product mixture was centrifuged to separate the immobilized trypsin beads from the digested protein solution. The supernatant was then extracted, C18 cleanup was performed with PepClean C18 spin columns (Pierce Biotechnology, Rockford, IL), and the resulting solution was diluted to the desired concentration (510 µM) in 50:50 H2O/MeOH. Peptides, Proteins, and Reagents. Peptides were obtained from Sigma Chemical (angiotensin I, bradykinin), Bachem (King of Prussia, PA) (val5-angiotensin I), and AnaSpec (San Jose, CA) (angiotensin II, phosphorylated angiotensin II, phosphorylated kinase domain of insulin receptor 4 [TRDIYETDYpYRK], PKA inhibitor substrate [GRTGRRNpSIHDIL], and phosphorylated protein kinase substrate-2 [KRpTIRR]). RS-Casein was obtained from Sigma Chemical. Immobilized TPCK treated trypsin was purchased from Pierce Biotechnology, Inc. RESULTS AND DISCUSSION As reported earlier, we have demonstrated that it is possible to selectively dissociate phosphorylated species in complex mixtures using direct infusion ESI-IRMPD-MS in a quadrupole ion trap.65 The results presented here extend this work to the direct analysis of peptide mixtures upon HPLC separation, allowing the selective dissociation of phosphorylated species following temporal resolution of analytes on a reversed-phase LC column. It should be emphasized that the selective dissociation of phosphorylated peptides is not due to a lower energy of activation but is instead due to a much greater IR absorption efficiency at 10.6 µm due to the phosphate chromophore. This issue was resolved in our previous study65 along with related work by Muddiman et al.22,25,28 in an FTICR instrument. For example, energy-variable collisional activated dissociation showed that doubly protonated angiotensin II and phosphorylated angiotensin II had similar critical energies, yet the irradiation time required to dissociate angiotensin II was 10-100 times greater than that needed for phosphorylated angiotensin II.65 This difference in photoabsorption efficiencies can be exploited to great advantage for selective differentiation of phosphorylated versus nonphosphorylated analogues.

LC-IRMPD-MS of an Eight-Peptide Mock Mixture. An LCIRMPD-MS experiment was performed on a mock mixture of model peptides, including angiotensin II and phosphorylated angiotensin II, to show that it is possible to perform selective dissociation on a complex mixture. An equimolar (10 µM) peptide mixture was prepared in 50:50 water/methanol and included phosphorylated protein kinase substrate-2 [KRpTIRR] (1), phosphorylated kinase domain of insulin receptor 4 [TRDIYETDYpYRK] (2), PKA inhibitor substrate [GRTGRRNpSIHDIL] (3), bradykinin (4), angiotensin II [DRVYIHPF] (5), phosphorylated angiotensin II [DRVpYIHPF] (6), val5-angiotensin I [DRVYVHPFHL] (7), and angiotensin I (8). A sample injection of 10 µL (100 pmol each) was loaded onto the column, and the peptides were eluted with a gradient (0.0-2.0 min 95% A, 2.0-2.5 min 9575% A, 2.5-42.5 min 75-50% A, 42.5-43.0 min 50-5% A, 43.053.0 min 5% A). The mass spectral data during the course of the chromatographic separation in the absence of laser irradiation was monitored to map the elution order and abundance of ions. The selected ion chromatograms (SICs) for each of the representative molecular ions were extracted, and the reconstructed ion chromatogram (RIC) that resulted from plotting each of the SICs on the same graph is illustrated in Figure 1A. In alternating scans throughout this same chromatographic run, the trapped ions were irradiated for 125 ms by the CO2 laser, producing tandem mass spectrometry data along with the previously discussed MS data. These data obtained from the LCIRMPD-MS experiment were plotted in the same way, as an RIC (Figure 1B). The intensity scale is the same in Figure 1A and B in order to allow direct comparison of each of the chromatograms. The peak areas of the unphosphorylated ions are reduced on the order of 5-20%; this can be observed for m/z 530, 524, 428, and 433. In contrast, following irradiation, the phosphorylated precursor ions are decreased from their initial intensity to the point where ion signal is no longer detected (i.e., their abundance is below the detection limit). The intensities of each of the nonphosphorylated peptides are slightly reduced from Figure 1A to B due to normal instrument fluctuation, by the extra step of 125-ms ion storage, and by a limited amount of dissociation of the all of the analyzed ions, as discussed in the next paragraph. Figure 2 shows the mass spectral data acquired across four of the eluting peaks, before (A-D) and after 125 ms of 10.6-µm irradiation (E-H). There are several instrumental variables that affect the relative peak areas of the eluting peptides when the reconstructed ion chromatograms from the MS and IRMPD-MS experiments are compared. For each step in the alternating scans, MS and IRMPDMS, an independent ion accumulation step is used, meaning that there is a certain degree of run-to-run error due to different numbers of accumulated ions. Replicate LC-MS experiments have shown that the run-to-run relative standard deviation for a given peak area ranges from 5 to 30%. The other variables that can affect ion abundances and thus peak areas are the extra 125-ms trapping step used in the IRMPD scan and, of course, the exposure of the trapped ions to IR radiation, both of which can lead to precursor ion loss. These two variables have been isolated and examined independently in back-to-back runs (n ) 4), and their contributions are insignificant when compared to the run-to-run variation due to ion accumulation from the ESI and ion-transfer events. Analytical Chemistry, Vol. 77, No. 17, September 1, 2005

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Figure 1. Eight-peptide mock mixture: reconstructed ion chromatograms for the selected peptide ions (A) without and (B) with laser irradiation (50 W, 125 ms).

Figure 2. (A-D) MS and (E-H) IRMPD-MS data taken at the indicated elution times. The representative precursor ions are labeled with an asterisk.

The first peptide eluted (in the dead volume) is KRpTIRR, and the corresponding mass spectrum shows the doubly and triply protonated species at m/z 455 and 303, respectively. Upon IR irradiation, both species dissociate via loss of H3PO4, a process that occurs in conjunction with dehydration for the triply charged peptide. The second peptide, TRDIYETDYpYRK, elutes at 14 min, and the corresponding mass spectrum is shown in Figure 2A. 5730


Exposure to IR irradiation results in dissociation of the triply protonated molecular ion, producing the b2, b4, b5, b6, and b8 fragment ions as well as the internal fragment TD (Figure 2E). The third peptide to elute is GRTGRRNpSIHDIL, with the triply protonated species detected at m/z 525. The dominant dissociation route upon IR exposure is the loss of H3PO4, resulting in an ion observed at m/z 492. The fourth and fifth peptides, bradykinin

Figure 3. RS1-Casein tryptic digest: reconstructed ion chromatograms for the selected peptide ions (A) without and (B) with laser irradiation (50 W, 125 ms).

and angiotensin II, are not phosphorylated. The ESI-mass spectra for these two peptides do not change upon irradiation and are dominated by doubly protonated bradykinin at m/z 530 and doubly protonated angiotensin II at m/z 524. The mass spectra obtained for angiotensin II before and after irradiation are shown in Figure 2B and F, respectively. The sixth peptide is phosphorylated angiotensin II, whose mass spectrum shows the doubly protonated species at m/z 564 (Figure 2C). Upon irradiation, it dissociates to form an abundant y2 ion along with y72+, b4, b5, b6, and the dephosphorylated y6 and b6 fragment ions (Figure 2G). The unidentified ion at m/z 530 in Figure 2C and G observed during elution of phosphorylated angiotensin II does not dissociate upon laser exposure, so it can confidently assigned as unphosphorylated. The last two peptides are val5-angiotensin I and angiotensin I. These peptides are detected as triply protonated species and do not dissociate upon irradiation; the mass spectra taken before and after irradiation of val5-angiotensin I are shown in Figure 2D and H, respectively. LC-IRMPD-MS of an rS1-Casein Tryptic Digest. After successful demonstration of the HPLC-IRMPD method for differentiation of phosphorylated and nonphosphorylated peptides in the mock mixture, the strategy was evaluated for mapping the phosphorylation states of peptides in a tryptic digest of a protein, RS1-casein. To obtain a pure sample of RS1-casein for LC-MS analysis, an RS-casein sample was subjected to preparative HPLC. The conditions used (outlined in the Experimental Section) allowed the separation of RS1-casein from RS2-casein and all other impurities. Following successful purification, tryptic digestion of the RS1-casein sample was performed, and the resulting peptide mixture was analyzed with LC-IRMPD-MS following removal of the digestion buffer with a PepClean C18 spin column. The massto-charge values of the tryptic peptides from RS1-casein identified

from direct infusion ESI-MS of the tryptic digest (data not shown) were targeted, and selected ion chromatograms were extracted from the raw data for the LC-MS and LC-IRMPD-MS experiments. All of the selected ion chromatograms were replotted together to create the reconstructed ion chromatograms displayed in Figure 3. The peptides have been labeled according to their order of elution; 20 tryptic fragments were identified in the experiment, as summarized in Table 1. Upon IR irradiation, several of the selected molecular ions vanish in Figure 3B, thus revealing their status as phosphorylated peptides. The peaks corresponding to the phosphorylated peptides are labeled with a circled P in Figure 3A and represent the phosphorylation of 46Ser, 48Ser, and 115Ser, three of the eight RS1-casein phosphorylation sites. No peptides were identified corresponding to the other five sites of phosphorylation, perhaps due to sample loss during C18 cleanup, poor ESI efficiency, nonstoichiometric phosphorylation, or a combination of these factors. Table 1 summarizes the m/z values of the 20 tryptic peptides, the sequences assigned based on ion m/z and primary sequence information, and the chromatographic peak areas without and with IR irradiation. There are some peak area fluctuations (positive and negative) between the LC-MS and LCIRMPD-MS data ranging approximately from 6 to 29% for all of the unphosphorylated ions monitored, due to the standard runto-run fluctuations in signal intensities. However, for the phosphorylated species (highlighted), the peak areas uniformly decrease by 96-100% of their initial intensities upon IR irradiation, thus allowing confident identification of their phosphorylation status. The 13th peptide that elutes, represented by m/z 586, is phosphorylated and also contains a bound sodium ion. This peak decreased in area from 8500 to 120 units, a net decrease of 99% peak area following irradiation. These data show that phosphoAnalytical Chemistry, Vol. 77, No. 17, September 1, 2005

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Table 1. Tabulated Reconstructed Ion Chromatographic Data from the LC-IRMPD-MS Analysis of an rS1-Casein Tryptic Digest elution order

nominal m/z

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

440 407 910 472 411 521 830 566 694 487 392 650 586 449 566 518 471 634 692 618

residues

primary sequence

phosphorylated

peak area MS

peak area IRMPD-MS

% intensity decrease

8-224+ 101-103(+Na)1+ 125-1321+ 35-422+ 91-1034+ 8-346+ 106-1192+ 106-1244+ 103-1193+ 101-1246+ 91-1055+ 37-584+ 101-119(+Na)4+ 101-1195+ 80-1025+ 80-1005+ 23-42(+MeOH)5+ 91-1002+ 23-342+ 104-1244+

HQGLPQEVLNENLLR LKK EGIHAQQK EKVNELSK YLGYLEQLLRLKK HQGLPQEVLNENLLRFFVAPFPEVFGK VPQLEIVPNpSAEER VPQLEIVPNpSAEERLHSMK KYKVPQLEIVPNpSAEER LKKYKVPQLEIVPNpSAEERLHSMK YLGYLEQLLRLKKYK VNELSKDIGsEsTEDQAMEDIK LKKYKVPQLEIVPNpSAEER LKKYKVPQLEIVPNSAEER HIQKEDVPSERYLGYLEQLLRLK HIQKEDVPSERYLGYLEQLLR FFVAPFPEVFGKEKVNELSK YLGYLEQLLR FFVAPFPEVFGK YKVPQLEIVPNSAEERLHSMK

N N N N N N Y Y Y Y N Y Y N N N N N N N

2500 840 480 1300 6500 2100 1000 4700 1300 4000 6000 9500 8500 3000 3100 4500 890 4600 1200 940

2800 1000 610 1200 7300 1900 0 180 0 110 4600 70 120 3200 2200 3800 670 3500 1500 740

-12 -19 -27 7.7 -12 9.5 100 96 100 97 23 99 99 -6.7 29 16 25 24 -25 21

Figure 4. Reconstructed ion chromatograms and the associated mass spectra of the selected precursor ions from the LC-IRMPD-MS analysis of a sample containing a 100 to 1 ratio of angiotensin II to phosphorylated angiotensin II (A) without and (B) with laser irradiation (50 W, 125 ms).

rylated species are selectively dissociated even if sodium-cationized rather than protonated. The 14th and 20th peptides are also interesting because they contain an unphosphorylated 115Ser residue, one which is phosphorylated in many of the other peptides monitored (peptides 7, 8, 9, 10, and 13). These two peptides, like all of the other unphosphorylated species, are not significantly affected by the irradiation event, which shows that this technique can be used to differentiate phosphorylation states 5732


on peptides with the same primary sequence; this can be used to examine the heterogeneity of a protein’s phosphorylation state. This set of experimental data thus demonstrates that it is possible to screen protein digests quickly and confidently for phosphorylated species based on whether ion abundance is decreased significantly upon exposure to short irradiation times using 10.6µm radiation, regardless of bound cationic species and protein to protein variation in phosphorylation.

Figure 5. Reconstructed ion chromatograms and the associated mass spectra of the selected precursor ions from the LC-IRMPD-MS analysis of a sample containing a 1 to 100 ratio of angiotensin II to phosphorylated angiotensin II (A) without and (B) with laser irradiation (50 W, 125 ms).

LC-IRMPD-MS of Stoichiometrically Unequal Peptide Mixtures. To demonstrate the effectiveness of LC-IRMPD-MS experiments for the selective dissociation of phosphorylated peptides in cases of nonstoichiometric concentrations of phosphorylated and unmodified peptides, a set of experiments were performed on mock mixtures of unphosphorylated and phosphorylated peptides in 100:1 or 1:100 molar ratios. The peptides monitored were angiotensin II and phosphorylated angiotensin II, allowing the observation of different degrees of phosphorylation of a single peptide to mimic what might be naturally observed in a cell extract. Figure 4 shows the reconstructed ion chromatograms, along with the associated mass spectra, resulting from the separation of a 100:1 mixture of unphosphorylated and phosphorylated angiotensin II, respectively. This is a simple model of the analysis one would expect in the case of 1% phosphorylation of a peptide, and from Figure 4A, it is apparent that it is possible to monitor ion signal from each of the peptides, m/z 524 (angiotensin II) and 564 (phosphorylated angiotensin II) as well as separate the two species chromatographically. Figure 4B is the reconstructed ion chromatogram obtained following irradiation of the ions formed from the eluting peptides, and it is obvious from this chromatogram as well as the mass spectra that the phosphorylated precursor ions are completely dissociated and the unphosphorylated ions remain intact. The chromatographic peak area for angiotensin II decreases by 2% during irradiation (83 800 to 82 100 units), whereas the peak area for phosphorylated angiotensin II decreases from 4500 to 17 units.

To demonstrate selective dissociation in a case where the phosphorylated species is present in excess, a 1:100 mixture of unphosphorylated and phosphorylated angiotensin II was subjected to LC-IRMPD-MS. The resulting reconstructed ion chromatograms and associated mass spectra in Figure 5 show that, upon irradiation, the phosphorylated peptide, despite being dominant in the LC-MS chromatograms, is completely dissociated; and only ion signal representing unphosphorylated angiotensin II remains. The mass spectra acquired over these chromatographic peaks show that, despite being present in excess, the phosphorylated peptide ion at m/z 564 is completely dissociated, forming the indicated fragment ions, while the abundance of the ion at m/z 524 ([Ang. II + 2H]2+) is largely unaffected by the irradiation event. The chromatographic peak area for angiotensin II decreases by 9% (3500 to 3200 units), whereas the peak area for phosphorylated angiotensin II decreases from 63 600 to 89 units upon irradiation. CONCLUSIONS Coupling LC-MS with IRMPD proves to be an effective strategy for efficient on-line screening of phosphorylation status of peptides in complex mixtures. Comparison of ion chromatograms with and without IR irradiation allows rapid pinpointing of the phosphorylated peptides based on significant reduction in their peak areas, as demonstrated for a mock mixture of peptides and a tryptic digest of RS1-casein. Unphosphorylated species are unaffected by the same degree of irradiation. The difference in IR absorption efficiency between phosphorylated and unphosphoAnalytical Chemistry, Vol. 77, No. 17, September 1, 2005

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rylated species is the basis for the notable response in the IRMPD experiments. The application of the LC-MS/IRMPD-MS technique to a tryptic digest of RS1-casein allowed differentiation of peptide phosphorylation states based on extent of dissociation upon exposure to 125 ms of 10.6-µm radiation. This ability to differentiate peptide phosphorylation state with this method is not significantly influenced by variables such as charge state, elution time, method of charging (protonation vs metal cationization), amino acid sequence (excluding modifications), or ion abundance. It was also demonstrated that it is possible to perform selective dissociation of phosphopeptides following LC separations in cases where there are 100:1 and 1:100 concentration ratios of phosphorylated versus unphosphorylated peptides, showing the potential utility

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of the LC-IRMPD-MS technique for the analysis of cell extracts in which nonstoichiometric degrees of phosphorylation would be expected. ACKNOWLEDGMENT Funding from the Welch Foundation (F1155) and the National Science Foundation (CHE-0315337) is gratefully acknowledged. The University of Texas Protein Analysis Facility is also gratefully acknowledged for their assistance in protein purification.

Received for review May 28, 2005. Accepted July 7, 2005. AC0509410

Differentiation of Phosphorylated and Unphosphorylated Peptides by

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