Selective, Sensitive, and Rapid Phosphopeptide Identification in

Anal. Chem. 2001, 73, 3305-3311

Selective, Sensitive, and Rapid Phosphopeptide Identification in Enzymatic Digests Using ESI-FTICR-MS with Infrared Multiphoton Dissociation Jason W. Flora and David C. Muddiman*,†,‡

Department of Chemistry, Virginia Commonwealth University, 1001 West Main Street, Richmond, Virginia 23284

Rapid screening for phosphopeptides within complex proteolytic digests involving electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR-MS) in the negative ion mode with infrared multiphoton dissociation (IRMPD) accompanied by improved phosphopeptide sensitivity and selectivity is demonstrated with the tryptic digests of the naturally phosphorylated proteins bovine r- and β-casein. All peptides in a complex proteolytic digest can be examined simultaneously for phosphorylation with a 4-s IR laser pulse at 7-11 W where phosphopeptide signature ions form upon irradiation, as the low energy of activation phosphate moiety cleavage transpires without the dissociation of the unphsophorylated peptide population. The tyrosine phosphorylated peptide HGLDN-pY-R, its nonphosphorylated analogue HGLDNYR, the kinase domain of insulin receptor unphosphorylated TRDIYETDYYRK, monophosphorylated TRDIYED-pY-YRK, and triphosphorylated TRDIpY-ETD-pY-pY-RK were also used as model peptides in this research. The sensitivity and selectivity of phosphopeptides is shown to greatly improve when the volatile base piperidine is used to adjust the pH of the ESI buffer, which results in greater than a 3-fold increase in relative ion abundance of a mono-phosphorylated peptide (HGLDNpY-R) over that of its unphosphorylated analogue. The addition of triethylamine provides comparatively equal ion intensities of modified and unmodified peptides, whereas ammonium hydroxide significantly suppresses phosphopeptide relative ion abundance when used to adjust the pH of the ESI buffer. With the addition of 20 mM piperidine in the ESI buffer, 30.6% of the amino acid sequence of the enzymatically digested β-casein, including the tetraphosphorylated tryptic fragment, was identified at a 500 pM (0.5 fmol/µL) concentration when electrosprayed at a rate of 3.3 nL/s. One of the most important regulatory mechanisms involved in gene expression and protein synthesis is the reversible * Corresponding author. Phone: 804-828-7510. Fax: 804-828-8599. Email: [email protected]. † Affiliate Appointment in Biochemistry and Molecular Biophysics. ‡ Massey Cancer Center. 10.1021/ac010333u CCC: $20.00 Published on Web 06/09/2001

© 2001 American Chemical Society

phosphorylation of the amino acids serine, threonine, and tyrosine.1,2 This covalent protein modification regulates cellular processes such as metabolism, growth, and reproduction.3-5 Subsequently, accurate determination of the site of phosphorylation provides valuable insight into the elucidation of many biological regulatory mechanisms.6,7 The standard biochemical approach to phosphoprotein mapping involves labeling the phosphate moiety with 32P, purification, and enzymatic digestion, followed by peptide separation via HPLC. These phosphopeptides are then subjected to Edman sequencing whereby the sites of phophorylation are identified by the radioactive labeling during the Edman cycles.8-12 Because this traditional biochemical technique can be laborious and unreliable,9 mass spectrometry (MS) has become an increasingly important approach for the accurate determination of the site of phosphorylation.13-18 Taking advantage of the relative speed, sensitivity, and adaptability of MS, phosphoproteins can be enzymatically digested, and each peptide of a known protein sequence can be screened for a phosphate group by the 80 Da mass increase.19,20 When the (1) Sun, H.; Tonks, N. K. Trends Biol. Sci. 1994, 19, 480-485. (2) Faux, M. C.; Scott, J. D. Trends Biol. Sci. 1996, 21, 312-315. (3) Krebs, E. G. Trends Biol. 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) Kennelly, P. J.; Krebs, E. G. J. Biol. Chem. 1991, 266, 15555-15558. (7) Songyang, Z.; Cantley, L. C. Trends Biol. Sci. 1995, 20, 470-475. (8) Boyle, W. J.; Geer, P. v. d.; Hunter, T. Methods Enzymol. 1991, 201, 110149. (9) Aebersold, R. H.; Watts, J. D.; Morrison, H. D.; Bures, E. J. Anal. Biochem. 1991, 199, 51-60. (10) Wettenhall, R. E.; Aebersold, R. H.; Hood, L. E. Methods Enzymol. 1991, 201, 186-199. (11) Wang, Y. H.; Fiol, C. J.; DePaoli-Roach, A. A.; Bell, A. W.; Hermodson, M. A.; Roach, P. J. Anal. Biochem. 1988, 174, 537-547. (12) Yan, J. X.; Packer, N. H.; Gooley, A. A.; Williams, K. L. J. Chromatogr. 1998, 808, 23-41. (13) Carr, S. A.; Hemling, M. E.; Bean, M. F.; Roberts, G. D. Anal. Chem. 1991, 63, 2802-2824. (14) Biemann, K. Biomed. Environ. Mass Spectrom. 1988, 16, 99-111. (15) Burlingame, A. L.; Boyd, R. K.; Gaskell, S. J. Anal. Chem. 1994, 66, R634R683. (16) Busman, M.; Schey, K. L.; John, E.; Oatis, J.; Knapp, D. R. J. Am. Soc. Mass Spectrom. 1996, 7, 243-249. (17) Cohen, P.; Gibson, B. W.; Holmes, C. F. B. Methods Enzymol. 1991, 201, 153-168. (18) Neubauer, G.; Mann, M. Anal. Chem. 1999, 71, 235-242. (19) Resing, K. A.; Johnson, R. S.; Walsh, K. A. Biochemistry 1995, 34, 9477. (20) Ladner, R. D.; Carr, S. A.; Huddleston, M. J.; McNulty, D. E.; Caradonna, S. J. J. Biol. Chem. 1996, 271, 7752.

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protein sequence is unknown, MS has also been employed for the identification of phosphorylated peptides within a proteolytic digest by observing the mass loss associated with the cleavage of the phosphate moiety induced by collisional activation dissociation (CAD)21,22 or enzymatically catalyzed dephosphorylation by treatment with phosphatases.23 Immobilized metal ion affinity chromatography (IMAC) of proteolytic digests prior to MS analysis for the selective micropreparative isolation of phosphopeptides has also been explored.24,25 In addition, electron capture dissociation of intact proteins has recently proven to be an effective method for phosphoprotein and phosphopeptide mapping of intact and digested phosphorylated proteins.26 These previously proposed MS techniques for phosphopeptide detection, albeit innovative improvements over the standard biochemical approach, require multiple steps or sample concentrations greater than 500 nM, which are often unavailable when dealing with real biological systems.18,27 Because of the biological significance of this cellular regulatory mechanism, strategies to selectively decrease the detection limit and analysis times of phosphorylated peptides are of great interest. However, the identification of these modified peptides remains challenging as a result of their low abundance in complex mixtures.27 Since the development of electrospray ionization,28 it has been a requisite tool when using mass spectrometry for the study of large biomolecules.29,30 The transfer of ions in solution into the gas phase gives ESI profound versatility by allowing the manipulation of solution conditions to enhance detection limits for target analytes. Oligonucleotides, typically electrosprayed in the negative ion mode, are best electrosprayed from a solution consisting of 60% acetonitrile, 20% isopropyl alcohol, and 20% 10 mM ammonium acetate with a final concentration of 20 mM piperidine and imidazole.31-37 Proteins and peptides are typically electrosprayed as positive ions using an electrospray buffer that consists of 50% methanol (or acetonitrile), the pH of which has been adjusted by the addition of acetic or formic acid. However, highly acidic (21) Huddleston, M. J.; Annan, R. S.; Bean, M. F.; Carr, S. A. J. Am. Soc. Mass Spectrom. 1993, 4, 710-717. (22) Carr, S. A.; Huddleston, M. J.; Annan, R. S. Anal. Biochem. 1996, 239, 180192. (23) Zhang, X.; Herring, C. J.; Romano, P. R.; Szczepanowska, J.; Brzeska, H.; Hinnebusch, A. G.; Qin, J. Anal. Chem. 1998, 70, 2050-2059. (24) Zhou, W.; Merrick, B. A.; Khaledi, M. G.; Tomer, K. B. J. Am. Soc. Mass Spectrom 1999, 11, 273-282. (25) Posewitz, M. C.; Tempst, P. Anal. Chem. 1999, 71, 2883-2892. (26) Stone, D.-H. S.; Hemling, M. E.; Carr, S. A.; Horn, D. M.; Lindh, I.; McLafferty, F. W. Anal. Chem. 2001, 73, 19-22. (27) Ogueta, S.; Rogado, R.; Marina, A.; Moreno, F.; Redondo, J. M.; Vazquez, J. J. Mass Spectrom. 2000, 35, 556-565. (28) Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse, C. M. Science 1989, 246, 64-71. (29) Smith, R. D.; Loo, J. A.; Edmonds, C. G.; Barinaga, C. J.; Udseth, H. R. Anal. Chem. 1990, 62, 882-899. (30) Mann, M.; Wilm, M. Trends Biochem. Sci. 1995, 20, 219-223. (31) Greig, M.; Griffey, R. H. Rapid Commun. Mass Spectrom. 1995, 9, 97-102. (32) Muddiman, D. C.; Cheng, X. H.; Udseth, H. R.; Smith, R. D. J. Am. Soc. Mass Spectrom. 1996, 7, 697-706. (33) Hannis, J. C.; Muddiman, D. C. Rapid Commun. Mass Spectrom. 1999, 13, 323-330. (34) Muddiman, D. C.; Null, A. P.; Hannis, J. C. Rapid Commun. Mass Spectrom. 1999, 13, 1201-1204. (35) Null, A. P.; Hannis, J. C.; Muddiman, D. C. Analyst 2000, 125, 619-625. (36) Hannis, J. C.; Muddiman, D. C.; Null, A. P. Advances in Nucleic Acid and Protein Analyses, Manipulation, and Sequencing; SPIE: San Jose, CA, 2000; pp 36-47. (37) Hannis, J. C.; Muddiman, D. C. Rapid Commun. Mass Spectrom. 1999, 13, 954.

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peptides often do not produce sufficient ion signals in the positive ion mode. Recent studies have demonstrated that peptides with a large number of acidic residues, which can be difficult to protonate, can be efficiently ionized in the negative ion mode, and dissociation of the deprotonated precursor ions provides useful structural information.38 Similarly, the increased acidity of phosphorylated peptides typically requires negative ionization for adequate ion signals.39,40 This research has explored electrospray buffers for the negative ion detection of phoshorylated peptides in the interest of achieving improved sensitivity and selectivity of this biologically significant posttranslational modification. The ultimate goal of this research is the development of a rapid identification method for phosphopeptides in proteolytic digests involving gas-phase infrared multiphoton dissociation (IRMPD)41 with electrospray ionization and Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR-MS). This technique exploits the fact that this posttranslational modification results in the formation of a covalent bond that will dissociate preferentially under low-energy dissociation techniques (e.g., IRMPD), producing signature product ions from which this modification can be identified.42 Analogous to CAD, IRMPD (at the appropriate laser power) results in signature ion formation involving the loss of H3PO4 (Mp, 98 Da) for phosphorylated serine and threonine or two product ions corresponding to the loss of H2O and the loss of HPO3 and H2O (M, 18 Da and M, 98) indicative of tyrosine dephosphorylation.16,42 Importantly, the approach demonstrated herein does not require ion isolation of each peptide prior to ion activation. With this technique, the entire proteolytic digest of a phosphoprotein can be interrogated for all sites of phosphorylation simultaneously in a single IRMPD experiment. EXPERIMENTAL SECTION Materials and Sample Treatment. The tyrosine phosphorylated peptide, HGLDN-pY-R, and the nonphosphorylated analogue, HGLDNYR [isoelectric point (pI) is 6.8], were obtained from New England Peptide, (Fitchburg, MA). This amino acid sequence corresponds to the gallus lysozyme tryptic fragment at amino acid positions 33-39. Kinase domain of insulin receptor unphosphorylated (TRDIYETDYYRK; pI ) 6.2), monophosphorylated (TRDIYED-pY-YRK), and triphosphorylated (TRDI-pY-ETD-pY-pY-RK) was purchased from ANASPEC (San Jose, CA). The sequence grade trypsin was purchased from Promega (Madison, WI). The bovine R- and β-casein, poly(ethylene glycol) with a 1000 Da average molecular weight (PEG-1000), acetonitrile, methanol, piperidine, triethylamine, and ammonium hydroxide were obtained from Sigma-Aldrich (St. Louis, MO), and all materials were used as received. Water was purified to 18 MΩ with a Barnstead Nanopure Infinity ultrapure water system. All pH measurements were made on 50-ml stock ESI buffers with a Mettler-Toledo (Hightstown, NJ) model MP220 pH meter prior to the addition of analyte. To avoid any matrix effects in the ESI process, bovine R- and β-casein were enzymatically digested with trypsin in 18 MΩ water (38) Ewing, N. P.; Cassady, C. J. J. Am. Soc. Mass Spectrom. 2000, 12, 105116. (39) Wilm, M.; Mann, M. Anal. Chem. 1996, 68, 1-8. (40) Wilm, M.; Neubauer, G.; Mann, M. Anal. Chem. 1996, 68, 527-533. (41) Little, D. P.; Speir, J. P.; Senko, M. W.; Oconnor, P. B.; McLafferty, F. W. Anal. Chem. 1994, 66, 2809-2815. (42) DeGnore, J. P.; Qin, J. J. Am. Soc. Mass Spectrom. 1998, 9, 1175-1188.

Figure 1. Single-acquisition ESI-FTICR mass spectra of three solution compositions at 100 nM showing the relative ion intensities of the phosphorylated peptide, Mp (HGLDN-pY-R) and the unphosphorylated peptide, M (HGLDNYR). A plot of the ratio of phosphorylated peptide intensity, IMp, to unphosphorylated peptide intensity, IM, is included. Each ratio represents the average of five spectra for each solution composition sequentially electrosprayed at 100 nM. The electrospray solutions were composed of 60% acetonitrile, 20% isopropyl alcohol, and 20% water with (A) pH adjustment to 10.9 with ammonium hydroxide, (B) 20 mM triethylamine (pH ) 11.1), and (C) 20 mM piperidine (pH ) 11.8).

at a pH of 7.3. The R- and β-casein and trypsin were at a molar ratio of 50:1, and the digestion was carried out at 37 °C for 24 h. 5 µL of ammonium hydroxide was added to the stock solution (200 µL) following enzymatic digestion to increase the pH (prior to dilution) and subsequently improve the solubility of the phosphopeptides, which may have precipitated during digestion. Mass Spectrometry and Protein Identification. All experiments were conducted using previously described microspray sources.43 ESI infusion was performed using a Harvard syringe pump, model PHD 2000. Infusion was carried out at a rate of 3.3 nL/sec through a 20-µL Hamilton syringe held at a potential of -2.5 keV. HGLDNYR, HGLDN(p)YR; kinase domain of insulin receptor (TRDIYETDYYRK) unphosphorylated, monophosphorylated, and triphosphorylated; and the R- and β-casein tryptic digests were electrosprayed at concentrations ranging from 500 pM to 100 nM, which are delineated in the text. HGLDN(p)YR and its unphosphorylated analogue were electrosprayed in 60% acetonitrile, 20% isopropyl alcohol, and 20% water whereas the Kinase domain insulin receptors, R-, and β-casein were electrosprayed from 50% methanol and 50% water. A modified Ionspec Corporation (Irvine, CA) FTICR-MS with a dual microelectrospray source44 was used in the negative ion mode, and all data involved two one-second hexapole accumulations followed by gated trapping.45 All spectra were single acquisitions and acquired with 512 k points at an ADC rate of 1 MHz and two zero fills unless otherwise noted. Therefore, all data was FT-limited by a 0.5-s time domain with an average resolution of ∼20 000 fwhm. Following electrospray ionization, hexapole accumulation,45 and gated trapping in the ICR cell, IRMPD41,46 was conducted on the trapped ions using a 25 W Synrad 48-2(W) (Mukilteo, WA) CO2 laser. The laser wavelength is 10.6 µm with a beam diameter of 3.5 mm. This laser is fitted with a 2.5× diffuser, which is directed through a BaF2 window into the ICR cell. A 1-µs “tickle” pulse is delivered at a 5 kHz clock frequency from the Synrad universal laser controller, preionizing the gas into a plasma state so that it

is just below the lase threshold prior to irradiation, resulting in predictable laser response. Peptide ions trapped in the ICR cell were irradiated for 4 s at powers ranging from 0 to 25 W. Internal calibration was accomplished using a dual electrospray source detailed elsewhere.44,47 The internal calibrant solution, PEG1000, was electrosprayed at 5 µM by the second ESI source. The PEG was accumulated in the hexapole for 500 ms and trapped in the ICR cell with the proteolytic β-casein digest. When internally calibrated IRMPD experiments were conducted, PEG ions were accumulated following IR laser irradiation of the tryptic fragments. Tryptic peptides and PEG ions were detected simultaneously where ion excitation/detection encompassed an m/z range of 200-5000 in 4 ms using a Chirp waveform with an excitation amplitude of 35 Vb-p. Database searching for the tryptic digests of R- and β-casein were conducted using the expert protein analysis system (ExPASy) proteomics server created by the Swiss Institute of Bioinformatics (SIB). Swiss-Prot release 30.14 and TrEMBL release 15.19 were the protein databases employed for all searches.48 RESULTS AND DISCUSSION Solution Composition for Selective and Sensitive Phosphopeptide Detection. Figure 1 shows three single-acquisition negative ion ESI-FTICR mass spectra of two peptides HGLDNpY-R, phosphorylated on the tyrosine, and its nonphosphorylated (43) Hannis, J. C.; Muddiman, D. C. Rapid Commun. Mass Spectrom. 1998, 12, 443-448. (44) Hannis, J. C.; Muddiman, D. C. J. Am. Soc. Mass Spectrom. 2000, 11, 876883. (45) Senko, M. W.; Hendrickson, C. L.; Emmett, M. R.; Shi, S. D.-H.; Marshall, A. G. J. Am. Soc. Mass Spectrom. 1997, 8, 970-976. (46) Peiris, D. M.; Yang, Y. J.; Ramanathan, R.; Williams, K. R.; Watson, C. H.; Eyler, J. R. Int. J. Mass Spectrom. Ion Processes 1996, 158, 365-378. (47) Flora, J. W.; Hannis, J. C.; Muddiman, D. C. Anal. Chem. 2001, 73, 12471251. (48) Bairoch, A.; Apweiler, R. Nucleic Acids Res. 2000, 28, 45-48.

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Figure 3. ESI-FTICR mass spectra of the tryptic digest of bovine β-casein at 500 pM. 10 acquisitions were averaged, and 30.6% of the sequence is covered by the labeled peaks. All tryptic fragments are isotopically resolved. Figure 2. Mass spectra of the kinase domain of insulin receptor, TRDIYETDYYRK, unphosphorylated, monophosphorylated, and triphosphorylated at equimolar concentration (100 nM) in a 50% methanol and 50% water ESI solution: (A) 20 mM triethylamine in the ESI solution (pH ) 11.0), and (B) ESI solution with 20 mM piperidine (pH ) 11.2).

analogue, HGLDNYR, at equimolar concentrations (100 nM) for when the ESI solution pH was adjusted by the addition of ammonium hydroxide (Figure 1A), triethylamine (Figure 1B), and piperidine (Figure 1C). Each bar in the inset graph represents the average ratio of the phosphopeptide to its unphosphorylated analogue for five spectra sequentially acquired from each solution composition under identical ESI conditions. Figure 1A shows the mass spectrum of the phosphorylated (Mp) and unphosphorylated (M) peptides electrosprayed for when the pH of the ESI solution was adjusted to 10.9 with ammonium hydroxide. The average ratio of the intensity of Mp to M using this ESI solution, plotted in the inset graph, A, is 0.40 ( 0.09 (all values are reported as the confidence interval of the mean at the 99% confidence level). Clearly, the phosphorylated peptide is underrepresented when the ESI solution contains ammonium hydroxide. Figure 1B shows a mass spectrum for when the pH was increased using 20 mM triethylamine (pH ) 11.1). Equal relative ion intensities are observed using this volatile base where the average ratio (inset graph, B) is 1.02 ( 0.22. Figure 1C shows the mass spectrum with selective phosphopeptide signal enhancement for when the pH is adjusted with 20 mM piperidine (pH ) 11.8). The phosphopeptide relative ion intensity is greatly improved when the ESI solution contains piperidine where the average ratio of Mp to M is 3.28 ( 0.33, which is plotted in the inset graph, C. It is important to note that relative ion intensities can vary on a daily basis and depend on a variety of factors; however, the trend in signal enhancement, discussed above, remains consistent for a given set of experiments. Another model peptide system was used to explore the relative signal enhancement of phosphopeptides using piperidine in the electrospray solution. Figure 2 shows single-acquisition ESI-FTICR mass spectra of the kinase domain of insulin receptor, TRDIYETDYYRK, unphosphorylated (M), monophosphorylated (Mp), and triphosphorylated (Mppp) at equimolar concentrations (100 nM). The mass spectrum in Figure 2A shows the relative ion intensity of the three peptide analogues with 20 mM triethylamine in the ESI solution where the most intense peak corresponds to 3308 Analytical Chemistry, Vol. 73, No. 14, July 15, 2001

the unphosphorylated species, with decreasing intensity as the number of phosphorylation sites increases on the peptide; however, for this amino acid sequence, the phosphopeptide relative ion intensities are selectively enhanced with 20 mM piperidine in the ESI solution, Figure 2B, with the triphosphorylated peptide as the dominant species. Phosphopeptide ion intensity enhancement by piperidine in the ESI buffer was also observed for the tryptic digests of the phosphorylated proteins bovine R- and β-casein electrosprayed at 10 nM where 76.4 and 67.9% of the amino acid sequences were observed, respectively (data not shown). Selective signal enhancement of phosphopeptides is achieved when the proteolytic digests are electrosprayed with 20 mM piperidine. The ESI-FTICR-MS negative ion sensitivity using the ESI buffer containing piperidine was investigated using the β-casein tryptic digest. Serial dilutions of the tryptic digest were electrosprayed with a blank ESI solution acquired after each sample concentration to obviate carryover. If weak or no signal was observed for a specific concentration, 10 acquisitions were averaged. Figure 3 shows a mass spectrum from the average of 10 acquisitions where the lowest concentration at which peptides from the β-casein tryptic digest were detected, 500 pM (0.5 fmol/µL). From mass spectra of either a single acquisition or the average of 10 acquisitions, 30.6% of the sequence was covered by the peptides detected at this concentration. On the basis of the results provided vida supra, all other peptides and protein digests discussed vida infra were electrosprayed in solutions containing 20 mM piperidine. Rapid Identification of Phosphorylated Peptides by IRMPD. The peptide HGLDNYR with and without phosphorylation on the tyrosine residue also provided a model system for the evaluation of ESI-FTICR-MS coupled with IRMPD for rapid phosphopeptide identification. These proteins were electrosprayed and trapped in the ICR cell at equimolar concentrations and irradiated over a wide range of laser power. Figure 4A shows the negative ion ESI-FTICR mass spectrum of these peptides without IR irradiation (note that this data set was acquired on a day different from that for the data shown in Figure 1, and therefore, the relative ion abundances are different). Figure 4B shows the spectrum of the phosphorylated and unphosphorylated peptides following infrared irradiation at 11.1 W for 4 s. The phosphorylated peptide loses both water and metaphosphoric acid, HPO3, upon irradiation, which is indicative of the dissociation pathway at low-

Figure 4. Negative ion mass spectra of HGLDNYR and HGLDN(p)YR at 100 nM concentration where Mp is the phosphorylated precursor ion and M is the unphosphorylated analogue: (A) spectrum without ion activation; (B) IR laser irradiation of the two peptides at 11.1 W for 4 s, where Mp dissociates by loss of water, Mp - H2O, as well as loss of water and metaphosphoric acid, Mp - H2O - HPO3 and M remains intact; (C) Plot of the relative ion intensity (ion amplitude/total ion amplitude) of M, Mp, Mp - H2O, and Mp - H2O - HPO3 as a function of IR laser irradiation energy (Watts). The diamonds and the cubes represent the unphosphorylated and the phosphorylated precursor relative ion intensities, respectively. The triangles and the circles represent the signature ions of phosphorylated tyrosine dissociation, water loss, and the concomitant loss of water and HPO3, respectively.

energy CID of peptides phosphorylated on tyrosine residues.42 At this laser power, the unphosphorylated peptide remains intact, but the phosphopeptide signature ions are observed because of the low energy of activation for the phosphate moiety loss.42 Figure 4C is a plot of relative ion intensity versus laser irradiation energy for the unmodified precursor ion (M), the phosphopeptide ion (Mp), and the signature ions corresponding to the neutral loss of water (Mp - H2O), and the concomitant loss of water and metaphosphoric acid (Mp - H2O - HPO3). Clearly, two important points are shown: (1) when irradiation energy increases, the phosphorylated peptide ion intensity decreases, but the intensity of the phosphopeptide signature ions, Mp - H2O and Mp - H2O - HPO3, increases. (2) as the irradiation power increases from 0 to 11.1 W, the relative intensity of the unphosphorylated peptide remains rather constant. This suggests that the IRMPD at the energies necessary to promote phosphate moiety loss do not affect the unmodified peptide analogue. Total ion intensity is reduced, and some low intensity sequence ions are observed above this laser power (11.1 W) at an irradiation time of 4 s.49 The kinase domain of insulin receptor (TRDIYETDYYRK) unphosphorylated, monophosphorylated, and triphosphorylated at equimolar concentrations (100 nM) was also used to study the effectiveness of IRMPD for the formation of phosphopeptide signature ions from singly and multiply phosphorylated species. Again, the phosphopeptides can be identified upon irradiation at the appropriate laser power and irradiation time without disruption of the unmodified peptide, M, providing rapid phosphopeptide detection in a single IRMPD experiment (data not shown). Peptides phosphorylated on the tyrosines provided the best model systems, because phosphotyrosines are known to be more stable than phosphoserines and phosphothreonines because of a lack of β-elimination pathways;50 therefore, IRMPD conditions that promote sufficient signature ion formation for a phosphotyrosine (49) Freitas, M. A.; Hendrickson, C. L.; Marshall, A. G. Rapid Commun. Mass Spectrom. 1999, 13, 1639-1642. (50) Affolter, M.; Watts, J. D.; Krebs, D. L.; Aebersold, R. Anal. Biochem. 1994, 223, 74-81.

peptides without dissociation of unmodified proteins will provide more than enough energy to promote signature ion formation from peptides with modified serines and threonines. The enzymatic digests of the phosphoproteins R- and β-casein were employed to further explore the identification of singly and multiply phosphorylated peptides using ESI-FTICR-MS coupled with IRMPD. Figure 5A shows the single-acquisition mass spectrum of the R-casein tryptic digest electrosprayed at 10 nM. The doubly charged peptide ion, m/z 974.5, represents the tryptic fragment containing amino acids 119-134 with the sequence of YKVPQLEIVPN-pS-AEER (one cleavage was missed by the tryptic digest after the lysine at position 120) where the amino acid 130, a serine, is phosphorylated (Mp). The multiply phosphorylated tryptic fragment (Mppppp), corresponding to positions 74-94 with the amino acid sequence QMEAE-pS-I-pS-pS-pS-EEIVPN-pS-VEQK is also observed at lower intensity. Serines 79, 81, 82, 83, and 90 are phosophorylated, and potential water loss is likely from one of five glutamic acids contained in the tryptic fragment. Figure 5B shows the result of IRMPD of the entire tryptic digest at an irradiation power of 11.1 W for 4 s. The unphosphorylated peptide fragments are unaffected by the infrared multiphoton irradiation, but the monophosphorylated fragment loses 98 Da (H2PO4), and the multiply phosphorylated peptide and its corresponding waterloss peak are removed from the spectrum. The signature ion, Mp - H2PO4 (amino acid sequence 119-134), is observed for the more intense monophosphopeptide; however, for the multiply phosphorylated peptide and its corresponding water-loss peak, IRMPD results in a distribution of five potential phosphoric acid losses, which are lost in the spectral noise due to low ion abundance. While the signature ion identifies the monophosphorylated peptide, the disappearance of the multiply phosphorylated peptide provides strong evidence for phosphorylation on that particular tryptic fragment as well. The tryptic digest of β-casein was also screened for phosphopeptides using negative ion ESI-FTICR-MS with IRMPD. Figure 6A shows the negative ion spectra of the β-casein proteolytic digest Analytical Chemistry, Vol. 73, No. 14, July 15, 2001

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Figure 5. ESI-FTICR-MS single-acquisition negative ion mass spectra of the tryptic fragments observed for R-casein (10 nM) where Mp represents the tryptic fragment with amino acid positions 119-134 phosphorylated on the serine at position 130. Mppppp represents the tryptic fragment containing positions 74-94 with phosphorylation at positions 79, 81, 82, 83, and 90. All identified amino acid positions are labeled, and 76.4% of the sequence is covered. The R-casein sequence is as follows (upper-case letters designate identified region of the amino acid sequence): mklliltclv avalarpkHP IKHQGLPQEV LNENLLRFFV APFPEVFGKe kvnelskdig sestedqame dikQMEAESI SSSEEIVPNS VEQKhiqkED VPSERYLGYL EQLLRlkkYK VPQLEIVPNS AEERlhsmke gihaqqkEPM IGVNQELAYF YPELFRQFYQ LDAYPSGAWY YVPLGTQYTD APSFSDIPNP IGSENSEKTT MPLW. (A) Spectrum without ion activation, (B) IR laser irradiation at 11.1 W for 4 s.

where Mpppp represents the tetraphosphorylated peptide corresponding to the amino acid positions 17-40 with phosphorylation of serines 30, 32, 33, and 34. Mp designates the singly phosphorylated peptide corresponding to the amino acid positions 48-63 with a phosphorylated serine at position 50. This doubly charged monophosphorylated peptide’s isotopic distribution overlaps the PEG oligomer at m/z 1030 (oligomer 23). All phosphorylation sites are observed for this modified protein, and upon infrared laser irradiation at 7.3 W for 4 s, Figure 6B, the unphosphorylated tryptic fragments remain intact while the phosphopeptides dissociate. The multiply phosphorylated peptide loses one water and 1 through 4 H2PO4 groups corresponding to each site of serine phosphorylation (siganture ions), whereas the low-intensity singly phosphorylated peptide is removed from the spectrum upon IR irradiation. For the multiply phosphorylated peptide, each signature ion is observed, indicating that the phosphopeptide detected by IRMPD contains a minimum of 4 phosphorylation sites. Internal calibration has been applied to the ESI-FTICR mass spectrum, Figure 6B, of the β-casein tryptic digest.44,47 Mass accuracies are listed for the mass spectrum acquired following laser irradiation and phosphate moiety loss. These accuracies 3310 Analytical Chemistry, Vol. 73, No. 14, July 15, 2001

range from -3.5 to 10.8 ppm. Albeit high-mass accuracy is not necessary for the identification of this tryptic digest, accurate mass measurement can play a significant role in database searching and protein identification. Accurate mass measurements on the order of (10 ppm nearly eliminates false positives when performing database searches.51 Furthermore, high-mass accuracies approaching peptide elemental composition determination can enable protein identity from a single tryptic peptide from which MS/MS sequence information has been obtained.51 CONCLUSIONS Negative ion detection of phosphopeptides using ESI-FTICR mass spectrometry, an area of great biological significance and interest, has been explored using three volatile bases for pH adjustment of the ESI solution. Clearly, the addition of piperidine to the ESI solution significantly enhances the phosphopeptide relative ion intensity, providing both sensitivity and selectivity to phosphopeptide analyisis when using electrospray ionization. Not only does up to a 3-fold increase in phosphorylated peptide relative (51) Clauser, K. R.; Baker, P.; Burlingame, A. L. Anal. Chem. 1999, 71, 28712882.

Figure 6. Internally calibrated ESI-FTICR-MS single-acquisition negative ion mass spectra of the tryptic digest of 10 nM bovine β-casein. Mpppp designates the tetraphosphorylated peptide corresponding to the amino acid positions 17-40 with phosphorylation of serines 30, 32, 33, and 34. Mp designates the singly phosphorylated peptide corresponding to the amino acid positions 48-63 with a phosphorylated serine at position 50. The amino acid positions of the identified tryptic fragments are labeled in the spectra and designated by upper-case letters below. 67.9% of the sequence is covered, with all sites of phosphorylation observed. The β-casein amino acid sequence: mkvlilaclv alalarELEE LNVPGEIVES LSSSEESITR inkkiekFQS EEQQQTEDEL QDKihpfaqt qslvypfpgp ipnslpqnip pltqtpvvvp pflqpevmgv skvkeamapk hkEMPFPKYP VEPFTESQSL TLTDVENLHL PLPLLQSWMH QPHQPLPPTV MFPPQSVLSL SQSKVLPVPQ KAVPYPQRDM PIQAFLLYQE PVLGPVRGPF PIIV. All poly(ethylene glycol) peaks used for internal calibration are labeled with their oligomer repeat number (italicized). All peaks are isotopically resolved. (A) Spectrum without laser irradiation where amino acid positions are listed with the charge state for each identified tryptic fragment. Mp overlaps the PEG oligomer 23, and this region of the spectra is expanded (each Mp isotope is labeled with a φ and each PEG isotope labeled with 23). (B) Laser irradiation at 7.3 W for 4 s. The multiply phosphorylated peptide loses one water and 1 through 4 H2PO4 (-1P, -2P, -3P, -4P) groups corresponding to each site of serine phosphorylation; this region of the spectrum has been expanded. The PEG oligomer 23 is expanded with no Mp ion present after IR laser irradiation. Experimental neutral monoisotopic masses are listed with mass errors listed in ppm. Asterisk (*), unidentified species. Two asterisks (**), mass accuracy is calculated from the most abundant isotope.

ion intensity over an unphosphorylated analogue occur, but also the detection of a 500 pM tryptic digest of bovine β-casein solution was enabled when piperidine was incorporated in the ESI buffer. With the enhanced phosphopeptide signal, IRMPD was employed for the formation of phosphopeptide signature ions upon irradiation. This research has demonstrated that within a complex protein digest, phosphopeptides can be simultaneously identified by a single IR laser irradiation event. Following this rapid phosphopeptide mapping strategy, sequencing can be accomplished by individual ion isolation and gas-phase dissociation of the target peptides of interest.

ACKNOWLEDGMENT The authors thank Dr. Yang-Sun Kim for her insightful discussions regarding this research. The authors gratefully acknowledge the financial support generously provided by the National Institutes of Health (R01HG02159) and the Department of Chemistry, Virginia Commonwealth University.

Received for review March 20, 2001. Accepted May 9, 2001. AC010333U

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