Phosphopeptide Analysis by Matrix-Assisted Laser Desorption Time-of

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Anal. Chem. 1996, 68, 3413-3421

Phosphopeptide Analysis by Matrix-Assisted Laser Desorption Time-of-Flight Mass Spectrometry Roland S. Annan* and Steven A. Carr

Department of Physical and Structural Chemistry, SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania 19406

In this paper we present methods for identifying and sequencing phosphopeptides in simple mixtures, such as HPLC fractions, at the subpicomole level by (+) ion matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-MS). Data are presented which indicate that when a reflectron time-of-flight mass spectrometer is used, MALDI can distinguish tyrosine phosphorylation from serine and threonine phosphorylation for peptides containing a single phosphate group. Phosphopeptides are identified in the (+) ion MALDI reflector spectrum by the presence of [MH - H3PO4]+ and [MH - HPO3]+ fragment ions formed by metastable decomposition. An abundant [MH - H3PO4]+ ion, accompanied by a weaker [MH - HPO3]+ ion indicates that the peptide is most likely phosphorylated on serine or threonine. In contrast, phosphotyrosine-containing peptides generally exhibit [MH - HPO3]+ fragment ions and little, if any [MH - H3PO4]+. Ambiguities do arise, most often with phosphopeptides that contain residues which readily lose water (such as unmodified serine), but these can often be resolved by recording a complete metastable fragment ion (postsource decay) spectrum. Postsource decay is shown here to be a viable technique for sequencing phosphopeptides. It can be used to distinguish between serine/ threonine and tyrosine phosphorylation and in many cases can be used to determine the exact site of phosphorylation in a peptide sequence. Nearly complete sequence coverage and phosphorylation site mapping is generally possible using ∼300 fmol of peptide. In the mid-1950s, phosphorylation was recognized as a reversible means of modulating protein function. By phosphorylating specific serine, threonine, and tyrosine residues, protein kinases alter the functions of their target proteins. Phosphorylated proteins are now known to play a critical role in transmitting extracellular signals to the nucleus. In fact, protein phosphorylation is probably the single most common intracellular signal transduction event. Among the thousands of proteins expressed in a typical mammalian cell, as many as one-third are now thought to be phosphorylated.1 Recent genomic sequencing results suggest that perhaps 4-5% of all eukaryotic genes code for protein kinases.1 The number of known protein kinases is increasing rapidly due, in large part, to gene cloning and sequencing techniques. Lagging behind the discovery of new protein kinases, however, is knowledge of their protein substrates. A universal * Corresponding author: e-mail, Roland S [email protected]. (1) Hubbard, M. J.; Cohen, P. Trends Biochem. Sci. 1993, 18, 172-177. S0003-2700(96)00221-1 CCC: $12.00

© 1996 American Chemical Society

recognition sequence has already been shown to not be applicable.2,3 Consensus sequences for particular protein kinases are, therefore, determined by comparing phosphopeptide sequences in known substrates. This approach is somewhat problematic when the substrates are unknown. Conversely, some clue as to the identity of the kinase responsible for phosphorylating a given protein is frequently provided by examining the amino acids bracketing a phosphorylated residue for a known recognition sequence. However, because most protein kinases are promiscuous, this approach is of limited utility for newly discovered protein kinases, even if they can be linked to a particular kinase family through sequence homology. The fact that a specific kinase can selectively phosphorylate a wide variety of proteins also leads to the question of how the enzyme recognizes specific residues from among the many hydroxyl groups found in the substrate protein. Although substantial progress has been made in understanding kinase recognition elements through the use of combinatorial peptide libraries,4,5 it is still necessary to reconcile the frequent exceptions found when an attempt is made to predict protein phosphorylation based on phosphorylation recognition sequences. All of these problems lead to the necessity for direct mapping of phosphorylation sites in proteins. Identifying phosphopeptides in protein digests usually requires labeling the protein with radioactive [32P]phosphate. Several rounds of HPLC purification may then be necessary to isolate a single peptide and prepare it for Edman sequencing. Unfortunately, the Edman chemistry itself is not particularly well suited to identifying phosphorylated residues. Each of the three major phosphoamino acids requires a different set of conditions under which sequencing by Edman chemistry will be successful. Both phosphoserine (pSer) and phosphothreonine (pThr) undergo β-elimination during Edman degradation to form PTH-dithioerythreitol byproducts.6,7 Conversion of pSer residues to S-ethylcysteine prior to Edman chemistry has been demonstrated to be a useful approach, especially when there are several serines in a (2) Kemp, B. E.; Pearson, R. B. Trends Biochem. Sci. 1990, 15, 342-346. (3) Aiken, A. Identification of Protein Consensus Sequences; Ellis Horwood Ltd.: Hemel Hempstead UK, 1990; pp 40-48. (4) Till, J. H.; Annan, R. S.; Carr, S. A.; Miller, W. T. J. Biol. Chem. 1994, 269, 7423-7428. (5) Songyang, Z.; Carraway, K. L.; Eck, M. J.; Harrison, S. C.; Feldman, R. A.; Mohammadi, M.; Schlessinger, J.; Hubbard, S. R.; Smith, D. P.; Eng, C.; Lorenzo, M. J.; Ponder, B. A. J.; Mayer, B. J.; Cantley, L. C. Nature 1995, 373, 536-539. (6) Mercier, J. C.; Grosclaude, F.; Ribadeau-Dumas, B. Eur. J. Biochem. 1971, 23, 41-51. (7) Meyer, H. E.; Hoffmann-Posorske, E.; Korte, H.; Heilmeyer, L. M. G. FEBS Lett. 1986, 204, 61-66.

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row.8 Unfortunately, this approach is not easily applied to pThr9 and is not applicable to phosphotyrosine (pTyr). Phosphotyrosine does not undergo β-elimination; however, the primary Edman product is not soluble in the typical extraction solvents. Even using modified sequencing conditions, under which the PTHphosphotyrosine is recovered in good yield, this amino acid does not provide an unambiguous signal under reverse phase HPLC conditions.10 The assignment of phosphorylation sites in peptides can also be made based on the release of 32P using several modified Edman sequencing protocols.11-13 Mass spectrometry is an analytical technique which has proven to be very useful in solving protein sequence problems. Since it is not hindered by the presence of posttranslational modifications or modified amino acids, it is an attractive alternative to conventional sequencing techniques. When the protein sequence is known, the molecular weight of a peptide may be used to corroborate the presence of phosphorylation by the 80 Da difference between the observed mass of the peptide and that calculated on the basis of the sequence. Any ambiguities can be resolved by sequencing the peptide using collision-induced dissociation (CID) tandem mass spectrometry (MS/MS).14,15 A mass spectrometry based approach to phosphopeptide analysis16-21 is especially powerful when used with electrospray ionization (ES),22 which has been shown to be more sensitive, by 1 order of magnitude, than fast atom bombardment, for both molecular weight measurements and peptide sequencing via MS/MS. Combined with on-line liquid chromatography, ESMS has formed the basis for several novel techniques for identifying phosphopeptides in protein digests without resorting to 32P labeling.23-25 Matrix-assisted laser desorption/ionization (MALDI) time-offlight mass (TOF) spectrometry26 is a relatively simple technique that has been characterized as extremely sensitive and somewhat tolerant of sample composition. For these reasons, MALDI (8) Meyer, H. E.; Hoffmann-Posorske, E.; Heilmeyer, L. M. G. In Methods in Enzymology, Protein Phosphorylation; Hunter, T., Sefton, B. M., Eds.; Academic Press: San Diego, 1991; Vol. 201, pp 169-185. (9) Dedner, N.; Meyer, H. E.; Ashton, C.; Wildner, G. F. FEBS Lett. 1988, 236, 77-82. (10) Meyer, H. E.; Hoffmann-Posorske, E.; Korte, H.; Donella-Deana, A.; Brunati, A. M.; Pinna, L. A.; Coull, J.; Perich, J. W.; Valerio, R. M.; Johns, R. B. Chromatographia 1990, 30, 691-695. (11) Roach, P. J.; Wang, Y. in: ref 8, pp 200-206. (12) Aebersold, R.; Watts, J. D.; Morrison, H. D.; Bures, E. J. Anal. Biochem. 1991, 199, 51-60. (13) Sullivan, S.; Wang, T. T. Anal. Biochem. 1991, 197, 65-68. (14) Biemann, K. In Methods in Enzymology, Mass Spectrometry; McCloskey, J. A., Ed.; Academic Press: New York, 1990; Vol. 193, pp 455-479. (15) Papayannopoulos, I. A. Mass Spectrom. Rev. 1995, 14, 49-74. (16) Poulter, L. ; Ang, S. G.; Gibson, B. W.; Williams, D. H. ; Holmes, C. F. B.; Cauldwell, F. B.; Pitcher, J.; Cohen, P. Eur. J. Biochem. 1988, 174, 497510. (17) Rossomando, A. J.; Wu, J.; Michel, H.; Shabanowitz, J.; Hunt, D. F.; Weber, M. J.; Sturgill, T. W. Proc. Natl. Acad. Sci.U.S.A. 1992, 89, 5779-5783. (18) Palczewski, K.; Buczylko, J.; Hooser, P. V.; Carr, S. A.; Huddleston, M. J. Crabb, J. W. J. Biol. Chem. 1992, 267, 18991-18998. (19) Hou, J.; McKeehan, K.; Kan, M.; Carr, S. A.; Huddleston, M. J.; Crabb, J. W.; McKeehan, W. L. Protein Sci. 1993, 2, 86-92. (20) Resing, K. A.; Johnson, R. S.; Walsh, K. A. Biochemistry 1995, 34, 94779487. (21) Ohguro, H.; Palczewski, K.; Ericsson, L. H.; Walsh, K. A.; Johnson, R. S. Biochemistry 1993, 32, 5718-5724. (22) Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse, C. M. Science 1989, 246, 64-71. (23) Huddleston, M. J.; Annan, R. S.; Bean, M. F.; Carr, S. A. J. Am. Soc. Mass Spectrom. 1994, 4, 710-717. (24) Affolter, M.; Watts, J. D.; Krebs, D. L.; Aebersold, R. Anal. Biochem. 1994, 223, 74-81. (25) Nuwaysir, L. M.; Stults, J. T. J. Am. Soc. Mass Spectrom. 1993, 4, 662-669. (26) Karas, M.; Hillenkamp, F. Anal. Chem. 1988, 60, 2299-2301.

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instrumentation has found its way into large numbers of protein biochemistry and core sequencing laboratories. Recently it was demonstrated that the energy-resolving characteristics of reflectron-type MALDI instruments make it possible to observe metastable fragment ions and record peptide fragment ion spectra in an experiment analogous to tandem mass spectrometry.27 Referred to as postsource decay (PSD),28 this technique produces predictable fragmentation along the peptide backbone. A few reports have been published showing the potential of this technique for peptide sequencing and glycopeptide analysis.29-32 The widespread use of MALDI mass spectrometers in biochemistry laboratories, and the current high level of interest in phosphorylation biology, combined with the difficulties associated with sequencing phosphopeptides by conventional techniques, led us to investigate the utility of MALDI for simplifying the process of protein phosphopeptide mapping. This article describes our experience utilizing (+) ion MALDI TOF and PSD for identifying and sequencing phosphopeptides isolated by HPLC fractionation of enzymatic digests. EXPERIMENTAL SECTION R-Casein was purchased from Sigma Chemical, the peptide AcRRLEIADpYAARG-amide was purchased from the University of Michigan Protein and Carbohydrate Facility, tyrosine phosphorylated calmodulin was prepared by in vitro reaction with purified insulin receptor catalytic domain as described previously,33 and rat osteopontin was prepared recombinantly from Chinese hamster ovary cells and purified in-house. Protein purity was confirmed by SDS-PAGE using 14% gels. The proteins were visualized with either Coomassie R250 (Bio-Rad) or Stains-All (Sigma). Phosphocalmodulin was digested with a combination of trypsin (Promega) and endoprotease Lys-C (Boerhinger Manheim) at a ratio of 25:1 (w:w, protein/enzyme) overnight in 100 mM NH4HCO3/2 mM EGTA at 38 °C. Otherwise, tryptic or endoprotease Asp-N (both from Boerhinger Manheim) digests were done at a ratio of 50:1 in 100 mM NH4HCO3 for 5-10 h at 38 °C. Phosphopeptides were purified by RP HPLC using a Michrom Ultrafast microprotein analyzer equipped with a 1 mm × 15 cm Reliasil C18 column. Peptides were eluted with water/acetonitrile/TFA gradients at 50 µL/min. Phosphopeptides from protein digests were isolated by LC/ESMS using the above HPLC conditions and a PE Sciex API III triple-quadrupole mass spectrometer. Voltage and scanning conditions were employed that favor the creation and detection of phosphopeptide specific marker ions.23 The eluent from the column is split 10:1 after the UV detector, with 5 µL/min going to the mass spectrometer, and 45 µL/min going to a Gilson fraction collector taking 1-min fractions. An R-cyano-4-hydroxycinnamic acid (R-HCA) (Aldrich) matrix was prepared by dissolving 10 mg in 1 mL of 50:50 ethanol/ (27) Tang, X.; Ens, W.; Standing, K. G.; Westmore, J. B. Anal. Chem. 1988, 60, 1791-1799. (28) Kaufmann, R.; Spengler, B.; Lutzenkirchen, F. Rapid Commun. Mass Spectrom. 1993, 7, 902-910. (29) Wu, Y.; Vath, J. E.; Huberty, M. C.; Martin, S. A. Anal. Chem. 1993, 65, 3015-3023. (30) Machold, J.; Utkin, Y.; Kirsch, D.; Kaufmann, R.; Tsetlin, V.; Hucho, F. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 7282-7286. (31) Huberty, M. C.; Vath, J. E.; Wu, Y.; Martin, S. A. Anal. Chem. 1993, 65, 2791-2800. (32) Wilm, M.; Houthaeve, T.; Talbo, G.; Kellner, R.; Mortensen, P.; Mann, M. In Mass Spectrometry in the Biological Sciences; Burlingame, A. L., Carr, S. A., Eds.; Humana Press: Totowa, NJ, 1995; pp 245-263. (33) Joyal, J. L.; Sacks, D. B. J. Biol. Chem. 1994, 269, 30039-30048.

acetonitrile. 2,5-Dihydroxybenzoic acid (DHB) (Fluka) was prepared by dissolving 10 mg in 1 mL of 50:50 ethanol/water. Samples were typically prepared by mixing 1-2 µL of an HPLC fraction with 1-2 µL of R-HCA matrix and spotting 0.5 µL of this solution on the target. Samples prepared in DHB are done the same way. Linear and reflectron MALDI mass spectra were recorded on a Micromass (Manchester, U.K.) TofSpec SE single-stage, gridless reflectron time-of-flight mass spectrometer with a maximum resolution in the reflector mode of ∼6000 (fwhm). The instrument has a 3.4-m effective path length and is coaxial in geometry. The three-element ion source provides a variable ion extraction region, useful for obtaining high fragment ion yields. In the reflectron mode of operation, the initial extraction voltage was set to 8.3 kV and the final accelerating voltage 25 kV. The reflector voltage is set to 28.5 kV. Samples are irradiated at a frequency of 5 Hz by 337-nm photons from a pulsed Laser Science (Cambridge, MA) nitrogen laser. Typically 20-50 laser shots are summed into a single mass spectrum. Spectra are calibrated externally using the monoisotopic MH+ ion from two peptide standards. Precursor ions for PSD are selected using a Bradbury-Nielsen-type ion gate,34 which has a resolution of ∼100. The drift region between the ion source and the reflector, which can also be referred to as the first FFR is 1 m. The vacuum in this region is typically 1.0 × 10-7 Torr. PSD spectra are typically recorded, under computer control, in either 8 or 10 segments, with each succeeding segment representing a 25% reduction in reflector voltage. Between 50200 laser shots are averaged per segment. Segments are joined and calibrated under computer control by the VG Opus data system. After the initial spectrum is reviewed weak segments can be rerecorded and added to, or substituted for, the original segments. Product ion resolution of >2000 (fwhm) has been achieved on this instrument, making monoisotopic mass determinations possible up to m/z 1800. This greatly reduces the uncertainty in assigning structures to ions. RESULT AND DISCUSSION The (+) ion linear and reflector MALDI-TOF spectra of the phosphoserine containing tryptic peptide VPQLEIVPNpSAEER (monoisotopic Mr ) 1659.7), isolated from R-casein S1 are shown in Figure 1. Both the linear and the reflector spectrum show an abundant [M + H]+ ion for the phosphopeptide. The reflectron spectrum, however, also displays ions representing dephosphorylated forms of the peptide, which are not observed in the linear spectrum. Because these ions are not observed in the linear spectrum, they must be fragment ions formed by the metastable decomposition of the phosphopeptide ion. The process of metastable decomposition in MALDI has been referred to as postsource decay.28 PSD is a process whereby a precursor ion that is sufficiently stable to be transported out of the ion source, but insufficiently stable to survive the flight to the detector, decomposes in the flight tube. It is believed that ions acquire excess internal energy necessary for fragmentation via multiple collisions, in the source, with matrix ions.35 When the precursor phosphopeptide ion decomposes in the flight tube, all of the fragment ions will have essentially the same velocity as the precursor but will have only a fraction of its kinetic energy. This (34) Bradbury, N. E.; Nielsen, R. A. Phy. Rev. 1936, 49, 388-393. (35) Spengler, B.; Kirsch, D.; Kaufmann, R. J. Phys. Chem. 1992, 96, 96789684.

Figure 1. MALDI spectrum of an HPLC fraction containing a serinephosphorylated peptide, VPQLEIVPNpSAEER: (A) linear mode using R-HCA; (B) reflectron mode using R-HCA; (C) reflectron mode using DHB. The peaks in the region 1300-1400 Da are from a coeluting nonphosphorylated peptide.

means that the precursor and metastable fragments will all strike the linear detector at the same time and will be detected at the same apparent mass. Thus metastable or postsource decay fragment ions are never observed in a linear spectrum. For this reason, the linear spectrum in Figure 1A shows only an [M + H]+ ion. A reflector-TOF is capable of discriminating fragment ions by flight time dispersion.27 Although all ions will have essentially the same velocity, the kinetic energy of a PSD fragment ion will be determined by the ratio of its mass to the mass of the precursor. Thus, the dephosphorylated peptide fragments, which have less kinetic energy than the phosphorylated precursor, do not penetrate as deeply into the reflector before they are turned around. As a result, the fragment ions have shorter flight times than the precursor and are therefore observed at lower masses than the precursor in the reflector spectrum. In Figure 1B, a percentage of the phosphopeptide ion population has decomposed in the flight tube, creating [MH - H3PO4]+ and [MH - HPO3]+ fragment ions. On the basis of the analysis of over 3 dozen phosphorylated peptides (data not shown) this series of ions is Analytical Chemistry, Vol. 68, No. 19, October 1, 1996

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Figure 2. MALDI PSD spectrum of the molecular ion cluster (monoisotopic MH+ ) 1660.1) from the serine-phosphorylated peptide VPQLEIVPNpSAEER. Fragment ions [yn - H3PO4]+ are indicated by the symbol yn2. This spectrum also contains an abundant b2-b7 ion series, as well as additional internal fragment ions, which have not been labeled for clarity.

generally characteristic of serine and threonine phosphorylated peptides. If these ions disappear when the spectrum is recorded in the linear mode, as seen in Figure 1A, one can be reasonably certain that the peptide is phosphorylated. The major fragment ion that is observed for serine and threonine phosphorylated peptides is [MH - H3PO4]+ or MH 98 Da. It is important to note that the [MH - H3PO4]+ fragment ion does not correspond to the dephosphorylated peptide but contains dehydroalanine in place of the phosphoserine residue. It is not, therefore, observed at the same mass as the nonphosphorylated peptide, but 18 Da lower. A second fragment ion, common to serine, threonine, and tyrosine phosphorylated peptides is[MH - HPO3]+ or MH - 80 Da. This ion is much less abundant than [MH - H3PO4]+ and is, of course, equivalent to the nonphosphorylated peptide. In the case of serine and threonine phosphorylation, this ion is always observed with the more abundant [MH - H3PO4]+ ion. Both the [MH - H3PO4]+ ion and the [MH - HPO3]+ ion will appear in the reflector spectrum at an m/z value less than that predicted by loss of 98 and 80 Da, respectively, because the mass calibration for the reflector is only accurate for ions with full accelerating energy. The presence of the nonphosphorylated peptide in the sample along with the phosphorylated peptide will complicate the interpretation of the reflector spectrum. However, interpretation of the linear spectrum will be straightforward, since it will contain only the [M + H]+ ion for each species. Thus, while it is frequently useful to record both the linear and reflector spectrum, it is essential to do so if one suspects that the nonphosphorylated peptide is present in the same HPLC fraction or sample as the phosphopeptide. With regards to phosphorylated peptides purified from protein digests, our experience has been that, for average tryptic peptides (8-15 residues), the nonphosphorylated peptide will usually elute in a later HPLC fraction. A feature of reflectron-type mass spectrometers, just recently developed, is the potential to sequence peptides.28 The same PSD process that gives rise to the loss of phosphate from phosphorylated peptides will fragment the peptide backbone in a predictable way. Recall that all fragment ions will have kinetic energies less than that of the precursor. Therefore, if the mass spectrometer is to detect these fragment ions at their correct masses, the reflector voltage must be lowered to bring these ions into energy 3416 Analytical Chemistry, Vol. 68, No. 19, October 1, 1996

focus. A complete PSD product ion spectrum can be acquired by successively lowering the reflector voltage, thereby bringing lower and lower mass fragment ions into focus at the reflecting detector, and recording the desired mass range in segments. The segments may be calibrated and assembled afterward. PSD spectra resemble low-energy CID spectra,36 similar to those commonly recorded on triple-quadrupole mass spectrometers. The spectra contain mainly bn and yn fragments with abundant internal fragments and low-mass immonium ions36,37 (for a review of peptide fragment ion nomenclature see ref 38). Since CID tandem mass spectrometry had already been shown to be a powerful tool for phosphopeptide sequencing,39-40 we hoped that PSD would prove equally valuable. PSD fragmentation typical of serine phosphorylated peptides is illustrated by the spectrum of VPQLEIVPNpSAEER (Figure 2). An ion gate34 with a resolution of ∼100 was used to select the MH+ precursor at m/z 1660.7 found in the spectrum shown in Figure 1B. If this was an unknown sequence, the large MH 80 ion (MH - HPO3) and even more abundant MH - 98 ion (MH - H3PO4) would strongly suggest that the phosphorylation site is on a serine or threonine residue rather than on tyrosine. Because the peptide has a C-terminal Arg, the spectrum is dominated by a yn series.41 An abundant y1 ion at m/z 175 is characteristic of peptides where the C-terminal residue is Arg. A nearly complete yn series up to y9, and the mass difference of 167 Da between y4 and y5, establishes the tenth residue as phosphoserine. The yn and the yn-H3PO4 (indicated as yn2) ion series account for all but 196 Da of the peptide molecular mass. The remaining mass can be accounted for only by PV or VP. Because trypsin rarely cleaves N-terminal to a proline residue, the sequence VP is suspected. This is confirmed by a series of internal fragments beginning with PQ and ending with PQLEI. The lack of a y6 ion in this spectrum is not unexpected, since we rarely (36) Rouse, J. C.; Yu, W.; Martin, S. A. J. Am. Soc. Mass Spectrom. 1995, 6, 822-835. (37) Carr, S. A.; Roberts, G.; Annan, R. S.; Hemling, M. E.; Hoyes, J. Proceedings 43rd ASMS Conference on Mass Spectrometry and Allied Topics, Atlanta GA, 1995; p 620. (38) Biemann, K. in: ref 14, pp 886-888, and references contained therein. (39) Gibson, B. W.; Cohen, P. in: ref 14, pp 480-501. (40) Michel, H.; Hunt, D. F.; Shabanowitz, J.; Bennett, J. J. Biol. Chem. 1988, 263, 1123-1130. (41) Martin, S. A.; Biemann, K. Int. J. Mass Spectrom. Ion Processes 1987, 78, 213-228.

Figure 3. Mass range expansion of two regions from the MALDI PSD spectrum of the serine phosphorylated peptide VPQLEIVPNpSAEER, shown in Figure 2.

observe a yn ion resulting from cleavage between a proline and the amino acid immediately C-terminal to it.42 The facile loss of neutral phosphate and/or phosphoric acid from bn and yn series fragment ions is characteristic of serine and threonine phosphorylated peptides and can sometimes make the assignment of the phosphorylated residue difficult. In this case, all of the y ions containing phosphoserine lose phosphoric acid to such an extent that the yn2 ion is more abundant than the precursor yn ion. Present to a lesser extent are ions corresponding to yn-HPO3. Unfortunately these ions appears at the same masses as nonphosphorylated yn ions. Taken together, the yn-HPO3 and yn-H3PO4 ions, in the absence of the precursor yn ion, might be mistakenly interpreted as yn and yn-H2O from a nonphosphorylated residue. The complete absence of a precursor yn ion is not uncommon, as is seen for the y10-y13 series in this spectrum. The difficulties this type of fragmentation presents when one attempts to sequence phosphopeptides, especially those containing multiple serine/ threonine residues, are discussed in greater detail below. Monoisotopic resolution of the product ions in this spectrum makes assigning ion structures to the m/z values more straightforward. An expansion of the baseline around m/z 784 (shown in Figure 3A) shows that a single fragment ion is present at m/z 784.2, whose mass corresponds to the y72 ion (calculated m/z 784.4) not the y6 ion (calculated m/z 785.3). Mass assignments in the PSD spectra are typically within 0.2-0.4 Da of the expected values. A similar expansion around m/z 882 (Figure 3B) indicates that there are two fragment ions present whose masses m/z 882.4 and 883.4, correspond to y7 (calculated m/z 882.4) and y82 (calculated m/z 883.4), respectively. These and other isotopic clusters of similar overall intensity in this mass region have isotope patterns close to theoretical, which helps to rule out the possibility that the appearance of this m/z 882-884 cluster is due to poor ion statistics. Side-chain cleavages resulting in the loss of H2O, HPO3, or H3PO4 from peptide fragment ions can significantly complicate the PSD spectrum of a phosphopeptide containing multiple serine or threonine residues. The PSD spectrum of ISHELESSpSSEVN (MH+ ) 1497.5), the C-terminal peptide of rat osteopontin, isolated from a tryptic digest by HPLC is shown, together with the PSD spectrum of the nonphosphorylated peptide, in Figure 4. This (42) Yu, W.; Vath, J. E.; Huberty, M. C.; Martin, S. A. Anal. Chem. 1993, 65, 3015-3023.

Figure 4. MALDI PSD spectrum of the molecular ion cluster: (A) from the serine-phosphorylated peptide ISHELESSpSSEVN (monoisotopic MH+ ) 1497.4); (B) from the nonphosphorylated peptide ISHELESSSSEVN (monoisotopic MH+ ) 1417.5). Fragment ions [bn - H3PO4 ]+ are indicated by the symbol bn2. Fragment ions labeled with an asterisk are the result of one or two H2O losses from the preceding bn or bn2 ion.

peptide contains five serine residues, and the phosphorylated residue is the third serine in a run of four. Both spectra exhibit a dominant bn ion series. This is typical of peptides that do not have a basic residue at the C-terminus. Characteristically, a bn series begins with an abundant b2 ion, except in those cases where a basic residue is present near the N-terminus. In this case, the bn series is initiated by the presence of the histidine at residue three. In the spectrum of the nonphosphorylated peptide, the signals for fragments b7-b11 are attenuated by losses of one and two water molecules from the serine residues at positions 7-10 (ions resulting from the loss of water are indicated by an asterisk). Nevertheless, the complete sequence of the peptide can be deduced from this spectrum. The bn ion series yields all but the first three N-terminal amino acids, but these can be determined from the mass differences between the y11, y12, and MH+ ions, and is further supported by several internal fragments whose masses fit the sequences SH, HE, and SHE. For the sake of clarity, not all of the ions have been labeled. In the phosphorylated peptide, loss of H3PO4 and/or water from this same series of bn ions has completely extinguished the signal for the precursor b9b11 ions. In the absence of the phosphorylated bn ion, it is impossible to tell whether the ions labeled with an asterisk are due to the loss of H3PO4 from a phosphorylated bn ion or are due to the loss of H2O from the bn ion of a nonphosphorylated serine. In the PSD spectrum of the nonphosphorylated analog, shown in Figure 4B, we observe that each nonphosphorylated serine residue gives rise to a bn ion and a bn - H2O, but never just the latter. We would predict the same pattern for each of the nonphosphorylated serines N-terminal to the phosphorylated residue in the spectrum of the phosphorylated peptide (Figure 4A). Indeed, the serines at positions 7 and 8 give rise to b7, b8, and bn - H2O ions, but the phosphoserine at position 9, yields only b9-H3PO4. Furthermore, all subsequent fragment ions that contain the phosphorylated Analytical Chemistry, Vol. 68, No. 19, October 1, 1996

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Figure 5. Reflectron MALDI spectrum of an HPLC fraction containing the tyrosine phosphorylated peptide DGNGpYISAAELR (monoisotopic MH+ ) 1345.6). The inset shows the expanded molecular ion region of the most abundant species present in this fraction. The distribution of isotopes and measured molecular weight indicate potentially extensive deamidation.Two other peptides are also present in this fraction.

serine residue lose H3PO4 to such an extent that little or no primary bn ions are observed, only abundant bn-H3PO4. This is shown by the b10 fragments in Figure 4A and more clearly by the y7-y12 series in Figure 2. We have observed this same fragmentation in the spectra of other phosphorylated and nonphosphorylated peptide pairs (data not shown). Taken together, these data strongly suggest the site of phosphorylation on this peptide is Ser9; however, this assignment is not completely unambiguous and points to the difficulty of assigning phosphorylation sites in peptides containing multiple serine/threonine residues. The reflectron spectrum of a phosphotyrosine-containing peptide, DGNGpYISAAELR (monoisotopic Mr ) 1344.6), isolated from a tryptic digest of in vitro phosphorylated bovine calmodulin, is shown in Figure 5. Phosphotyrosine-containing peptides fragment to lose HPO3 (MH - 80) as discussed above. However, since phosphotyrosine is much more stable than either phosphoserine or phosphothreonine, this ion is normally not very abundant. The loss of H3PO4 (MH - 98), which is usually quite abundant in the case of peptides phosphorylated on serine and threonine, is not favored in the case of a phosphotyrosinecontaining peptide, since it requires cleavage of a bond adjacent to an aromatic ring, forming a radical cation. The loss of HPO3 from the phosphotyrosine side chain, followed by the loss of H2O from somewhere else on the peptide, can give rise to a pronounced [MH - 98]+ ion, especially in peptides that contain multiple serine or threonine residues, since both of these amino acids readily fragment to lose H2O (see Figure 7). In general, however, we have observed that the reflectron spectra of tyrosine phosphorylated peptides rarely display the [MH - 98]+ ion. Thus it should be possible to distinguish tyrosine from serine and threonine phosphorylation based on the type of fragment ions present. For example, the phosphopeptide shown in Figure 5 contains both serine and tyrosine residues. The lack of an abundant MH - 98 ion in the reflectron spectrum, however, suggests strongly that the peptide is tyrosine phosphorylated. The PSD spectrum of the phosphotyrosine-containing peptide, DGNGpYISAAELR, is shown in Figure 6. The [MH - HPO3]+ ion is again much more abundant than the [MH - H3PO4]+ ion, 3418 Analytical Chemistry, Vol. 68, No. 19, October 1, 1996

suggesting that this peptide is tyrosine phosphorylated. Confirmation of this comes from the presence of a phosphotyrosine immonium ion found at m/z 216. This ion is present in the spectra of all of the phosphotyrosine peptides we have examined and is frequently very intense. We have not observed an immonium ion for either phosphoserine or phosphothreonine in a PSD spectrum. Localization of phosphotyrosine residues is simplified relative to the identification of phosphoserine, because the loss of HPO3 from peptide fragment ions is not favored. Thus, while the 87 Da mass difference between y5 and y6 indicates an unmodified serine, the mass difference of 243 Da between y7 and y8 unambiguously assigns the tyrosine at residue 5 as the phosphorylated residue. Ions resulting from the side-chain cleavage of HPO3 are usually only observed when accompanied by a very abundant bn or yn ion (see for instance the y9 ion). Even though this peptide contains a C-terminal arginine residue, and no basic residue near the N-terminus, the PSD spectrum also contains a nearly complete bn series beginning with b2 and ending at b10. The mass difference between b4 and b5 confirms the assignment of the fifth residue as phosphotyrosine. The measured molecular mass of this peptide was greater than the calculated value by 1.3 Da. In this mass range, our mass measurement accuracy is usually (0.2 Da. The isotope profile of the MH+ ion shown in the inset of Figure 5 suggests that the 1-Da mass difference is due to partial deamidation of the peptide. Evidence from the PSD spectrum confirmed this. The mass of the b3 ion, and the mass difference between y9 and y10, both indicated that the asparagine at position 3 had been converted to aspartic acid. It is well-known that an Asn residue in the Asn-Gly sequence context is highly susceptible to deamidation.43 This determination was made possible by the high resolving power of the mass spectrometer, for both precursor and product ions. In the case of a tyrosine-phosphorylated peptide that contains several residues capable of losing water, it may be difficult to rule out serine or threonine phosphorylation, based solely on the fullenergy reflectron spectrum. This is illustrated in Figure 7 by the reflectron spectrum of the tyrosine-phosphorylated peptide TRDIYETDpYYRK (monoisotopic Mr ) 1701.8), a partial tryptic peptide from the insulin receptor catalytic domain. The presence of a pair of fragment ions, [MH - HPO3]+ and [MH - H3PO4]+, with the latter ion being more abundant, suggests, incorrectly, that this peptide is serine or threonine phosphorylated. The PSD spectrum of this peptide, however, rules out this possibility (Figure 8). Because this peptide contains arginine residues near both the Nand C-termini, both bn and yn series ions are present, although only a few of the bn ions have been labeled for clarity. The abundant immonium ion at m/z 216 suggests a phosphorylated tyrosine residue. The presence of the b3, b6, and b8 ions at the expected masses rules out phosphorylation at either of the threonines and eliminates the possibility of phosphorylation at the tyrosine residue at position 5. The unusually abundant b3 and b8 ions can be ascribed to favored cleavage C-terminal to an aspartic acid residue.42 Unfortunately, the lack of a y3 ion in this spectrum prevents one from distinguishing between phosphorylation of Tyr9 or Tyr10. The side-chain fragmentation of the molecular ion (shown expanded in the inset of Figure 8) is dominated by [MH - HPO3]+ and [MH - H3PO4]+ ions in a ratio similar to that observed in the reflectron spectrum (Figure 7). The loss of water from the molecular ion of tyrosine phosphorylated peptides is (43) Geiger, T.; Clarke, S. J. Biol. Chem. 1987, 262, 785-794.

Figure 6. MALDI PSD spectrum of the molecular ion cluster (monoisotopic MH+ ) 1346.9) from the tyrosine phosphorylated peptide DGNGpYISAAELR. Fragment ions [yn - HPO3]+ are indicated by the symbol yn2. Fragment ions labeled with an asterisk are the result of H2O loss from the preceding ion. Deamidation of the asparagine to aspartic acid is indicated by (N f D).

Figure 7. Reflectron MALDI spectrum of the tyrosine phosphorylated peptide TRDIYETDpYYRK (monoisotopic MH+ ) 1702.7).

generally only observed in peptides that contain several serine, threonine, or to a lesser extent tyrosine residues. Thus the unusually large ion at MH - 98 may be explained as a combination of MH - H3PO4 and MH - HPO3 followed by the loss of H2O from the other threonine and/or tyrosine residues. Although we have examined only a limited number of multiply phosphorylated peptides, in all cases (serine and tyrosine phosphorylation) the reflector spectra showed sequential losses of neutral phosphate, phosphoric acid, water, and ammonia. Figure 9 shows the reflector and linear (inset) spectra of the triply phosphorylated peptide from the insulin receptor described above. In addition to the potentially complex fragment ion pattern observed in the reflector spectra of multiply phosphorylated peptides, the sensitivity with which the data can be acquired is greatly reduced, because the ion current is distributed over many different species. As is obvious from this example, recording a linear spectrum for multiply phosphorylated peptides is essential to making some sense of the data, since in the linear mode, all of the fragment ions from an individual species will revert to a single MH+ ion. The inset shows the presence of two MH+ ions, the major species corresponding to the triphosphorylated species. The mass of the small peptide corresponds to the diphosphorylated species. The ratio of MH - H3PO4 and MH - HPO3 fragment ions to parent ion is matrix dependent. R-HCA yields the most intense

fragment ions, while DHB gives the least as is shown in Figure 1B,C. We have not recorded spectra of phosphopeptides using hydroxypicolinic acid,44 in spite of its reported ability to reduce postsource decay,45 because in our hands it is very much less sensitive than either R-HCA or DHB for peptide analysis. In the range of 300-1500 fmol, the extent of side-chain fragmentation from the molecular ion seems to have little dependence on the laser irradiance. The ratio of intact phosphopeptide to phosphorylated side-chain fragment ion remained rather constant, no matter how energetically the sample is irradiated. This is probably because even threshold irradiance provides sufficient energy for this facile fragmentation pathway. In practice, spectra are always recorded using the lowest possible laser irradiance, since under these conditions, resolution will be the highest. For serine, and probably threonine phosphorylated peptides, however, the ratio of intact phosphopeptide to phosphorylated side-chain fragment ion does appear to be sequence dependent, since some peptides yielded almost entirely metastable fragment ions, while others give abundant phosphopeptide molecular ions (data not shown). We have found that we can reduce fragmentation by lowering the initial field strength of the acceleration region, but, unfortunately, this also degrades the overall resolution.37 It is also likely that the final accelerating voltage, the length of the flight tube, and the residual gas pressure in the instrument will affect the degree of fragmentation. CONCLUSIONS Only a few of the hundred or even thousand amino acids that compose a typical phosphoprotein sequence are phosphorylated. Phosphopeptide mapping, therefore, requires that the researcher identify a few phosphopeptides from among the many other nonphosphorylated peptides in a protein digest. This is typically accomplished by HPLC fractionation of radioactive 32P-labeled protein digests, followed by Cherenkov counting of the HPLC fractions. Recently it has become possible to do this without resorting to radioactivity, by using LC/ESMS techniques which (44) Wu, K. J.; Steding, A.; Becker, C. H. Rapid Commun. Mass Spectrom. 1993, 7, 142-146. (45) Karas, M.; Bahr, U.; Strupat, K.; Hillenkamp, F.; Tsarbopoulos, A.; Pramanik, B. N. Anal. Chem. 1995, 67, 675-679.

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Figure 8. MALDI PSD spectrum of the molecular ion cluster (monoisotopic MH+ ) 1702.7) from the tyrosine phosphorylated peptide TRDIYETDpYYRK. Fragment ions [yn - HPO3]+ are indicated by the symbol yn2. The inset shows an expansion of the spectrum in the region where fragments resulting from side-chain fragmentation of the molecular ions are found.

Figure 9. Reflectron MALDI spectrum of the triply tyrosine phosphorylated peptide TRDIpYETDpYpYRK (monoisotopic MH+ ) 1862.9) showing multiple losses of phosphate and water. The inset shows a spectrum of the same sample recorded in the linear mode and indicates that a small amount of diphosphorylated peptide may be present in the sample.

take advantage of in-source CID to produce phosphate specific marker ions.23,46 Nevertheless, these HPLC fractions frequently contain more than one peptide and often contain many peptides. Metastable decomposition and prompt fragmentation of phosphopeptides has been described previously, using a variety of mass spectrometers as a way to distinquish phosphopeptides from nonphosphorylated peptides. Gibson and Cohen reported observing fragmentation of phosphopeptides in (+) ion FAB spectra recorded on a magnetic deflection mass spectrometer.39 Substantial loss of H3PO4 from the molecular ion of a serinephosphorylated peptide occurs during (+) ion MALDI-ion trap analysis.47 Previously, Talbo and Mann48 demonstrated that (46) Ding, J. M.; Burkhart, W.; Kassel, D. B. Rapid Commun. Mass Spectrom. 1994, 8, 94-98. (47) Jonscher, K. R.; Yates, J. R. Proceedings 41st ASMS Conference on Mass Spectrometry and Allied Topics, San Francisco CA, 1993; p 695a-695b. (48) Talbo, G.; Mann, M. In Techniques in Protein Chemistry; Crabb, J. W., Ed.; Academic Press: San Diego, 1994; Vol. 5, pp 105-113.

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sulfated peptides could be distinguished from phosphorylated peptides on the basis of their (-) ion MALDI reflectron spectra. However, in these experiments, tyrosine-, serine-, and threoninephosphorylated peptides all gave essentially identical reflectron spectra, showing an ion at [M - H - 97]-. Griffin and co-workers recently reported observing extensive fragmentation in the (+) ion reflectron MALDI spectrum of a serine-phosphorylated peptide from β-casein.49 We have presented here a simple method that is well suited for identifying phosphopeptides in these HPLC fractions that relies on MALDI TOF. Phosphopeptides can be identified in (+) ion MALDI reflectron spectra based on the presence of characteristic fragment ions. Confirmation of a phosphopeptide can be achieved by rerecording the spectrum in the linear mode. This technique has low-femtomole sensitivity, and our results suggest that it may be generally possible to distinguish tyrosine phosphorylation from serine/threonine phosphorylation, at least for singly phosphorylated peptides based on the type of fragment ions present. Any ambiguity can, as demonstrated, usually be cleared up by recording a full PSD spectrum. The evidence presented here indicate that PSD is a viable technique for sequencing phosphopeptides. It can be used to distinguish between serine/threonine and tyrosine phosphorylation and, in many cases, can be used to determine that exact site of phosphorylation in a peptide sequence, although, as demonstrated, this becomes very much more difficult as the number of serines and threonines and phosphoserines/phosphothreonines increases. Several chemical modifications have been reported for converting the highly labile phosphoserine to another more stable residue.20,50 These have been demonstrated to be useful for mass spectrometry based methods of sequencing and thus would no doubt benefit the determination of multiple phosphoserine sites by PSD as well. A major advantage of PSD decay as a sequencing technique is the small amount of material required to record a high-quality (49) Giffin, P. R.; MacCoss, M. J.; Eng, J. K.; Blevins, R. A.; Aaronson, J. A.; Yates, J. R. Rapid Commun. Mass Spectrom. 1995, 9, 1546-1551. (50) Cohen, P.; Gibson, B. W.; Holmes, C. F. B. in: ref 14, pp 153-168.

spectrum. All of the PSD spectra shown in this report were recorded on between 300 and 1000 fmol of peptide. We can routinely acquire analytically useful PSD spectra on 200 fmol, and some sequence information can usually be determined on very much less than this (data not shown). We almost always record the PSD spectrum from the same sample spot that was used to record the reflectron and the linear spectrum. In fact, the PSD spectrum shown in Figure 2 was recorded, using the same sample spot, 2 months after the reflector spectrum was acquired. The extent of fragmentation we have observed in the (+) ion reflectron mode is dependent primarily on the matrix used and the strength of the initial acceleration region. Many older reflectron instruments either have single-stage acceleration regions or have no control over the voltages that define the initial acceleration region. Therefore, the extent of the fragmentation described above may vary in different MALDI instruments. The recent refinement of “delayed extraction” techniques applied to a

MALDI-TOF mass spectrometer51 may prove useful for limiting the amount of side-chain fragmentation occurring in serine- and threonine-phosphorylated peptides. Based on a concept developed by Wiley and McLaren in 1953,52 this technique, which has been shown to greatly enhance resolution, would also probably limit PSD by lessening the amount of collisional activation occurring in the source.

(51) Vestal, M. L.; Juhasz, P.; Martin, S. A. Rapid Commun. Mass Spectrom. 1995, 9, 1044-1050. (52) Wiley, W. C.; McLaren, I. H. Rev. Sci. Instrum. 1953, 26, 1150-1157.

AC960221G

ACKNOWLEDGMENT The authors acknowledge the other members of the Research MS group at SmithKline, most especially Messrs. Michael Huddleston and Gerald Roberts, and Professor David Sacks of Brigham and Womens Hospital and Harvard Medical School. We are also greatful to Dr. John Hoyes from Micromass for helpful discussions regarding PSD calibration. Received for review March 7, 1996; Accepted July 9, 1996.X

X

Abstract published in Advance ACS Abstracts, August 15, 1996.

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