Laser-Induced Dissociation of Phosphorylated Peptides Using

09_Cotter 10/19/07 7:57 AM Page 133

Laser-Induced Dissociation of Phosphorylated Peptides Using Matrix Assisted Laser Desorption/Ionization Tandem Time-of-Flight Mass Spectrometry Dongxia Wang,1 Philip A. Cole,2 and Robert J. Cotter2,*

1Biotechnology Core Facility, National Center for Infectious Disease, Center for Disease Control and Prevention, Atlanta, GA; 2Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, MD

site or concensus sequence is present in a given Abstract tryptic peptide. Here, we investigated the frag- Reversible phosphorylation is one of the mentation of phosphorylated peptides under most important posttranslational modifications laser-induced dissociation (LID) using a of cellular proteins. Mass spectrometry is a MALDI-time-of-flight mass spectrometer with a widely used technique in the characterization of curved-field reflectron. Our data demonstrated phosphorylated proteins and peptides. Similar that intact fragments bearing phosphorylated to nonmodified peptides, sequence information residues were produced from all tested peptides for phosphopeptides digested from proteins can that contain at least one and up to four be obtained by tandem mass analysis using phosphorylation sites at serine, threonine, or either electrospray ionization or matrix assisted tyrosine residues. In addition, the LID of laser desorption/ionization (MALDI) mass phosphopeptides derivatized by N-terminal spectrometry. However, the facile loss of neutral sulfonation yields simplified MS/MS spectra,

phosphoric acid (H3PO4) or HPO3 from precur- suggesting the combination of these two types sor ions and fragment ions hampers the precise of spectra could provide an effective approach determination of phosphorylation site, particu- to the characterization of proteins modified by larly if more than one potential phosphorylation phosphorylation.

*Author to whom all correspondence and reprint requests should be addressed: Robert J. Cotter, Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, MD. E-mail: [email protected].

133 09_Cotter 10/19/07 7:57 AM Page 134

134 ______Wang, Cole, and Cotter

Key Words: Phosphorylation; MALDI; time-of- expense of other pathways that would produce flight mass spectrometry; curved-field reflectron; sequence ions; particularly those that retain the N-terminal sulfonation; fragmentation. phosphate group, so that it is not possible to fix Introduction the actual position of the phosphate on the peptide sequence. In tandem instruments which uti- Reversible phosphorylation is one of the lize low energy CID, that is: quadrupole ion most common and important regulatory mod- traps, hybrid quadrupole/time-of-flight mass ifications of cellular proteins (1). This modifi- spectrometers, Fourier transform mass spec- cation plays a crucial role in maintaining and trometers, and so on, ions are initially cooled regulating protein structure and function in a through collisions with inert gas in an RF ion number of biological processes and signal guide, retaining little of the internal energy transduction pathways. Among 6000 yeast pro- derived from the ionization process as they teins recently reported in the genomic database enter the mass analyzer. Following precursor of budding yeast Saccharomyces cerevisiae, about ion selection multiple low-energy collisions 250 are protein kinases that are responsible for increase the internal energy slowly, favoring substrate phosphorylation (2). Phosphorylation fragmentation of the weaker bonds that leads usually occurs on one of three amino acid not only to the facile loss of anionic groups residues: serine, threonine, or tyrosine. The (such as phosphate) and other posttranslational majority of phosphorylation occurs on serine modifications, but also to preferential cleavages residue (~90%), with only a small number of at proline, aspartic acid, and glutamic acid phosphoproteins containing phosphothreonine residues (8,9). (~10%) and phosphotyrosine (~1%). The iden- Electron capture dissociation was introduced tification and characterization of phosphory- recently as a fragmentation method for Fourier lated proteins is a major effort in proteomics. transform mass spectrometers (10–12) and has Mass spectrometry has become a powerful shown considerable promise as an activation tool for protein identification, providing rapid method for elucidating phosphorylation sites and reliable peptide sequencing using a number (13–15). For peptides (and for proteins in so- of fragmentation methods. The determination of called top down methods [16]) electron capture phosphorylation sites within modified proteins, dissociation produces predominant c and z. (or however, remains a challenge. This is primarily c. and z) fragment ions resulting from the cleav- because the most common method used to age of the amine backbone bond of precursor induce fragmentation in electrospray ionization ions. Fragmentation is more distributed, rather (ESI) and matrix assisted laser desorption/ion- than selective for specific residues or side ization (MALDI) mass spectrometers, low- groups, and, therefore, an abundance of energy collision-induced dissociation (CID), sequence-specific fragment ions are available results in significant loss of neutral phosphoric for the determination of both sequences and

acid (H3PO4 98 Da) or HPO3 (80 Da) from phos- the location of posttranslational modifications. phorylated amino acid residues (3–7). The facile Similarly, it has also been suggested that loss of these groups could result from either MALDI mass spectra obtained on time-of-flight gas-phase β-elimination of the phosphate ester mass spectrometers produce charge remote frag- from phosphoserine or phosphothreonine mentation, both from post-source decay and CID residues, or from a two-step process involv- processes (17). Similar to the high-energy CID

ing the loss of HPO3 followed by the loss of processes described previously for sector mass H2O from phosphotyrosine residues. This spectrometers (18), fragmentation is not local- fragmentation pathway is generally at the ized at specific sites, but well-distributed and

Clinical Proteomics ______Volume 2, 2006 09_Cotter 10/19/07 7:57 AM Page 135

Laser-Induced Dissociation of Phosphorylated Peptides ______135

therefore more informative of structure. The RELEELNVPGEIVEpSLpSpSEESITR, were pur- tandem time-of-flight (TOF/TOF) mass spec- chased from Sigma (St. Louis, MO). α-Cyano- trometer provides a particularly effective instru- 4-hydroxycinnamic acid was from Aldrich mental configuration for observing charge (Milwaukee, WI). The synthetic peptides, remote fragmentation of MALDI ions with better RLEpSR and RLEpTR, were prepared by the mass selection than the single analyzer instru- Synthesis and Sequencing Facility at the Johns ments for which post-source decay was initially Hopkins University School of Medicine (Balti- developed (19). Unlike most other tandem and more, MD). Peptides VSSDGHEpYIYVDP- hybrid instruments in which precursor ions are MQLPY and VSSDGHEpYIpYVDPMQLPY collisionally cooled, the internal energy result- were gifts from Dr. Akhilesh Pandey. All other ing from the initial MALDI processes can be peptides were synthesized with FMOC chem- used to drive the fragmentation. Such laser- istry by solid phase peptide synthesis and induced dissociation (LID) processes can also purified by reverse-phase high-performance be complemented by the inclusion of a collision liquid chromatography. chamber to produce single, high energy collisions that also lead to charge remote fragmen- Sulfonation tation. The motivation for this current work was Phosphopeptides were derivatized with the possibility that well-distributed fragmentation SPITC as previously described (21). In brief, could also be observed for phosphorylated pep- the reagent solution was prepared by dissolv-

tides and could provide sequence-specific ing SPITC (10 mg/mL) in 20 mM NaHCO3 fragments that were more informative for deter- (pH ~ 9.0). The sulfonation reaction was car- mining phosphorylation sites. ried out in a 0.6-mL Eppendorf tube by mixing Here, we present our investigation of the 9 µL of reagent solution with 1 µL of peptide fragmentation of phosphorylated peptides by solution (approx 10–100 pmol). After incuba- LID using a MALDI-TOF/TOF mass spectrom- tion for 30 min at 55°C the reaction was ter- eter with a curved-field reflectron (20). Our minated by adding 1 µL of 1% trifluoracetic data demonstrate that intact fragments bearing acid (TFA). The sample was then loaded onto phosphorylated residues could be produced a micropipet tip (C18 OMIX, Varian, Lake from a series of peptides with from one to four Forest, CA), washed with 3 × 10 µL of 0.1% phosphorylation sites at serine, threonine, or TFA and followed by eluting with 10 µLof tyrosine residues; and that these could be used 75% acetonitrile/0.1% TFA. The solution was effectively to map the phosphorylation sites. In evaporated to dryness by SpeedVac and then µ addition, we utilized a method developed resuspended with 10 L of ddH2O. recently in our laboratory (21) to form N-terminal sulfonate derivatives of phosphopeptides, Mass Spectrometry which led to far simpler and more interpretable All MS and MS/MS spectra were acquired mass spectra that could be used to complement in the positive ion mode using a Kratos Ana- the direct fragmentation sequencing. lytical (Manchester, UK) AXIMA-CFR high performance mass spectrometer equipped Experimental with a pulsed extraction source, a 337-nm pulsed nitrogen laser and a curved-field Materials reflectron. The AXIMA is in effect a tandem All chemicals used in this study were of configuration (Fig. 1) that has been used analytical grade. 4-Sulfophenyl isothiocyanate both in a LID (Fig. 1A) and high energy CID (SPITC), sodium bicarbonate and the peptide, mode with the inclusion of a collision chamber

Volume 2, 2006 ______Clinical Proteomics 09_Cotter 10/19/07 7:57 AM Page 136

136 ______Wang, Cole, and Cotter

Fig. 1. Schematic of the AXIMA CFR mass spectrometer using (A) laser-induced dissociation or (B) collision- induced dissociation.

(Fig. 1B) (20). In this study, only the LID were also observed. This well-distributed mode was used. The matrix solution was pre- fragmentation, including both sequence ions pared by dissolving α-cyano-4-hydroxycin- and phosphate loss, is consistent with the namic acid (10 µg/µL) in 50% acetonitrile suggestion by Gross et al. (17) that there is containing 0.1% of TFA. A thin layer of the considerable charge-remote fragmentation in matrix was applied onto the sample plate first, the post-source dissociation processes that followed by the addition of 0.5 µL of sample occur following laser desorption. and 0.5 µL of matrix and was allowed to dry Figure 2 shows the fragmentation of three at room temperature. peptides (FQpSEEQQQTEDELQDK, RLEpTR, Results and Discussion and VSSDGHEpYIYVDPMQLPY) containing a single phosphorylated residue. For the two Ten phosphopeptides were investigated peptides with phosphoserine and phospho- and their fragmentation results are summa- threonine (Fig. 2A,B) neutral loss of phos- rized in tabular form (Table 1). These pep- phoric acid from the molecular ion is the tides include phosphorylation on all three dominant process, whereas fragments result-

target residues, serine, threonine, and tyro- ing from the loss of both H3PO4 and HPO3 sine, at various positions and contain up to from the precursor ion of the phosphotyrosine- four phosphorylated residues within individ- containing peptide (Fig. 2C) were observed ual species. The masses of the protonated but not dominant. At the same time, there is peptides range from 740.3 to 3123.6 Da. As extensive sequence-specific (b- and y-series) listed in Table 1, all peptides tested produced fragmentation that includes intact sequence nearly complete sets of b- and y-series fragments retaining the phosphate group, and sequence ions including these containing sequence fragments showing the loss of 98 U intact phosphorylated amino acid residues, (in the case of serine and threonine) or 80

while the loss of HPO3 (–80 Da) and H3PO4 mass units (in the case of tyrosine). In Fig. 2A, (–98 Da) from some precursors and fragments the sequence ions y14 and y15 all include the

Clinical Proteomics ______Volume 2, 2006 09_Cotter 10/19/077:57AMPage137

Table 1 Product Ions in the MALDI MS/MS Spectra of Phosphorylated Peptides Product ions Exp. Calc. Peptides M+ H+ M+H+ Mass Type Mass Type Mass Type Mass Type Mass Type

SApSEPSLHR 1063.5 1063.9 175.6 y1 425.3 y3 738.0 y6 906.2 y7* 983.3 MH-80

312.2 y2 609.1 y5 807.9 y7-98 965.3 MH-98

FQpSEEQQQTEDELQDK 2061.8 2062.0 390.8 y3 977.7 y8 1430.9 b11* 1620.8 y13 1916.3 y15*

503.5 y4 1106.1 y9 1461.6 b12-98 1673.4 b13* 1965.1 MH-98

632.5 y5 1234.3 y10 1491.6 y12 1689.9 y14-98 1983.2 MH-80

747.0 y6 1315.5 b10* 1560.0 b12* 1787.6 y14*

876.4 y7 1362.3 y11 1575.3 b13-98 1818.5 y15-98

LPKINRSApSEPSLHR 1784.9 1784.8 174.9 y1 721.9 b6 1063.3 y9* 1348.5 y12-98 1686.5 MH-98

312.2 y2 738.6 y6 1079.0 b10-98 1447.3 y12* 1705.3 MH-80

339.9 b3 808.3 b7 1096.5 b10-80 1513.8 b14-98 1767.7 b15*

425.6 y3 880.2 b8 1121.8 y10-98 1531.5 b14-80

452.3 b4 906.2 y7* 1177.0 b10* 1576.0 y13* 137 566.3 b5 965.6 y9 -98 1219.5 y10* 1611.1 b14*

609.4 y5 976.3 y8* 1333.5 y11* 1670.1 b15-98

CLRRNpSDR 1099.5 1099.8 175.4 y1 529.2 b4 727.0 y5* 845.8 b7-80 1001.3 MH-98

290.4 y2 571.5 y4* 729.7 b6-80 883.7 y6* 1082.1 b8*

373.3 b3 643.4 b5 810.6 b6* 925.2 b7*

456.7 y3* 712.3 b6 -98 827.7 b7-98 984.1 b8-98

RLEpSR 740.3 740.3 157.3 b1 269.7 b2 468.5 b4-98 584.5 y4* 723.1 b5*

175.3 y1 341.9 y2 * 486.6 y4-98 625.7 b5-98

243.9 y2-98 399.2 b3 566.1 b4* 642.2 MH-98

RELEELNVPGEIVE - 3123.6 3123.9 477.0 y4 885.2 b7 1479.4 b13 1958.2 (b17-98)* 2750.1 MH-3_ pSLpSpSpS EESITR 98-80 × × 528.0 b4 902.2 y7 1609.3 b14 2027.2 b18-2 98 2830.1 MH-3 98 × 605.8 y5 983.4 b8 1678.3 b15-98 2044.0 y16**** 2847.9 MH-2 98-80 × 657.2 b5 1137.2 b10 1790.8 b16-98 2124.7 (b18-98)* 2927.9 MH-2 98 × 735.0 y6 1266.4 b11 1860.3 b17-2 98 2140.6 y17**** 2945.9 MH-98-80 × 771.2 b6 1379.7 b12 1888.8 b16* 2732.4 MH-4 98 3025.8 MH-98

(Continued) 09_Cotter 10/19/07 7:57 AM Page 138 * * * 8 4 16 * 17 1098.9 b Mass Type Mass * 1916.4 b * 1916.4 MH-98 * 2095.8 * * 6 -80 MH-80 6 7 -98 13 15 -80)* -98 598.2 y 5 4 16 16 * 2012.6 b ** 14 16 820.9 b 482.2 b 1835.2 2113.7 * 1542.1 b * b * 656.2 MH-98 * 656.2 b * 934.9 ** 2175.8 MH-98 ** 2175.8 MH-80 ** 2193.7 2 5 -80 1802.3 b 4 5 -98 639.1 b -98 639.1 -80 741.8 y 10 12 -80)* 1915.7 (b 14 15 4 5 12 12 Product ions Product * 1674.3 b ** 1995.9 b 11 12 580.1 b 705.8 b 269.7 b * 500.4 y * 670.8 y * 1231.1 b * 1365.1 b (b * 1445.7 ** 1754.3 b ** 1882.1 b 1 4 3 2 -80 b 4 8 9 9 -80 625.7 b 11 10 b 4 10 -80 1330.0 b -80)* 1526.0 b 9 10 356.1 y 543 b 954.7 b 157.0 1311.9 b 1311.9 1068.1 b 1068.5 b able 1. (Continued) T * 462.6 b * 462.6 * 574 y * 1411.0 b 1 3 6 3 6 6 2 3 8 -98 399.3 b -80 b b b 2 8 b 987.8 b 754.3 1231.6 (b 7 7 955.0 b 747.7 y 747.9 y 410.8 y 175.3 y 445.5 b 874.5 1150.9 1445.1 874.5 1150.9 258.3 y Mass Type Mass Mass Type Type Mass Type Mass Type + 712.1 b 712.2 b M + H + H Exp. Calc. 1116.4 1116.6 1116.6 1116.4 375.7 b * Fragments containing one phosphorylated residue. ** Fragments containing two phosphorylated residues. **** Fragments containing four phosphorylated residues. pY MAPYDNY VSSDGHE pY IYVDPMQLPY- 2193.6 2194.2 583 VSSDGHE pY I VDPMQLPY- 2273.9 2273.7 584.0 Peptides M + RLE pT R 754.4

138 09_Cotter 10/19/07 7:57 AM Page 139

Fig. 2. MALDI MS/MS spectra of the peptides: (A) FQpSEEQQQTEDELQDK, (B) RLEpTR, and (C) VSSDGHEp- YIYVDPMQLPY. pX represents phosphorylated serine, threonine, or tyrosine residue. Asterisks label the intact product ions containing phosphate group.

139 09_Cotter 10/19/07 7:57 AM Page 140

140 ______Wang, Cole, and Cotter

Fig. 3. MALDI MS/MS spectra of the peptides: (A) VSSDGHEpYIpYVDPMQLPY and (B) RELEELNVPGEIVEp- SLpSpSpSEESITR.

phosphate group, while the ions y5 to y13 do but there is no difficulty in determining which not, suggesting that the phosphate is located one of three tyrosine residues is phosphory-

at the serine residue. In addition, there is also lated (Fig. 2C) by comparing the b7 and b8 a y14-98 ion; and the ions b10 to b13 include ions, and noting that all ions from b8 to b16 phosphate, consistent with the location of the include phosphate. phosphate group toward the N-terminus. In A peptide may carry more than one phos- the spectrum of the peptide RLEpTR (Fig. 2B), phorylated residue (Fig. 3). Where these are all the phosphothreonine residue can be identified phosphotyrosine residues, identification of the

by comparing the masses between y1 and y2 modification sites is less problematic because ions or between b3 and b4 ions. The peptide neutral losses are not dominant as we suggested VSSDGHEpYIYVDPMQLPY is relatively large in the discussion of Fig. 2C. Fragmentation

Clinical Proteomics ______Volume 2, 2006 09_Cotter 10/19/07 7:57 AM Page 141

Laser-Induced Dissociation of Phosphorylated Peptides ______141

Fig. 4. (continued)

of the peptide VSSDGHEpYIpYVDPMQLPY sequence-specific fragmentation; and the irregu- (Fig. 3A) reveals that two internal but not the lar removal of these groups from fragments can C-terminal tyrosine residues are modified by further complicate the spectra. The peptide, phosphorylation. This is determined by the RELEELNVPGEIVEpSLpSpSpSEESITR, con-

mass differences between b7 and b8 and between tains five serine residues with four of them b9 and b10 ions; and the observation of several modified by phosphorylation. In addition to the large b ions (b10 to b16) containing two modified parent ion, the mass spectrum (Fig. 3B) was residues validates the identification. In con- dominated by several high mass fragments trast, the analysis of multiple phosphoserine- resulting from the consecutive release of one to containing peptides is more challenging four phosphoric acids from the precursor ion. because the elimination of one or more phos- At the same time, other product ions were phoryl groups from the molecular ion are dom- observed in the mass range from 450 to 2200 Da

inant fragmentation channels that compete with with high signal/noise ratio. The detection of y6

Volume 2, 2006 ______Clinical Proteomics 09_Cotter 10/19/07 7:57 AM Page 142

142 ______Wang, Cole, and Cotter

Fig. 4. MALDI MS/MS spectra of (A) N-terminally derivatized RLEpSR, (B) underivatized RLEpSR, (C) N- terminally derivatized LPKINRSApSEPSLHR, and (D) underivatized LPKINRSApSEPSLHR. Peptides were N-terminal derivatized with 4-sulfophenyl isothiocyanate as described.

and y7 ions, containing no mass increase by in the positive ion mode, as b-series (and other phosphate group, suggests that the serine N-terminal) products are neutralized or nega- residue at the fourth C-terminal position is not tively charged by the presence of the sulfonic

modified whereas the presence of b16, y16, and acid group. To determine the usefulness of the y17 indicates that all other four serines were sulfonation approach for phosphopeptides, we phosphorylated. derivatized the peptide, RLEpSR, with SPITC N-terminal sulfonation has been used to at its N-terminus and examined the fragmen- simplify MS/MS spectra for de novo peptide tation of the peptide derivative. The MS/MS sequencing (21,22). Modification of the spectrum (Fig. 4B) of the native peptide shows N-terminus of a peptide by a sulfonic acid that its fragmentation yielded product mix-

group leads to the formation of y-series ions tures including b (b1 to b4) and y (y1, y2, and y4)

Clinical Proteomics ______Volume 2, 2006 09_Cotter 10/19/07 7:57 AM Page 143

Laser-Induced Dissociation of Phosphorylated Peptides ______143

ions, and some neutral loss ions (b4-98, y2-98, Institute (Jennifer VanEyk, PI). Mass spectral and y4-98). In contrast, the fragmentation of analyses were carried out at the Middle the derivatized peptide (Fig. 4A) generated Atlantic Mass Spectrometry Laboratory. exclusively y-series ions in the range from 100 to 600 Da, and no N-terminal ions, indicating References that N-terminal sulfonation is also capable of 1. Hunter, T. (2000) Signaling: 2000 and beyond. directing the fragmentation of phosphorylated Cell 100, 113–127. peptides. In addition to the loss of phosphate 2. Goffeau, A., Barrell, B. G., Bussey, H., et al. from the molecular ion, we also observe the (1996) Life with 6000 genes. Science 274, 546–567. additional characteristic ions corresponding to + + 3. Annan, R. S. and Carr, S. A. (1996) Phospho- MH -tag and MH -tag-98. These characteristic peptide analysis by matrix-assisted laser des- ions are also observed for the spectrum of orption time-of-flight mass spectrometry. Anal. the N-terminal sulfonated derivative of the Chem. 68, 3413–3421. peptide LPKINRSApSEPSLHR (Fig. 4C). In 4. Busman, M., Schey, K. L., Oatis, J. E., and contrast to the mass spectrum of the under- Knapp, D. R. (1996) Identification of phosphorylation sites in phosphopeptides by positive ivatized phosphopeptide (Fig. 4D), which and negative mode electrospray ionization- shows both b- and y-series ions, the simple tandem mass spectrometry. J. Am. Soc. Mass mass spectrum of the derivatized peptide Spectrom. 7, 243–249. provides relatively easy location of the phos- 5. DeGnore, J. P. and Qin, J. (1998) Fragmenta- phorylation site. tion of phosphopeptides in an ion trap mass spectrometer. J. Am. Soc. Mass Spectrom. 9, 1175–1188. 6. Tholey, A., Reed, J., and Lehmann, W. D. (1999) Conclusions Electrospray tandem mass spectrometric stud- We investigated the fragmentation behav- ies of phosphopeptides and phosphopeptide analogues. J. Mass Spectrom. 34, 117–123. iors of phosphorylated peptides using 7. Moyer, S. C., Cotter, R. J., and Woods, A. S. MALDI-TOF mass spectrometry. Our results (2002) Fragmentation of phosphopeptides by demonstrated that LID allows the formation atmospheric pressure MALDI and ESI/ion of intact fragments containing phosphorylated trap mass spectrometry. J. Am. Soc. Mass Spec- residues and provides important information trom. 13, 274–283. for localizing phosphorylation sites. This over- 8. Yu, W., Vath, J. E., Huberty, M. C., and Martin, S. A. (1993) Identification of the facile gas- comes a major problem in analyzing phos- phase cleavage of the Asp-Pro and Asp-Xxx phopeptides that is the neutral loss of H3PO4 peptide bonds in matrix-assisted laser desorp- and HPO3 from tandem mass spectra prod- tion time-of-flight mass spectrometry. Anal. ucts. Combining with N-terminal sulfonation Chem. 65, 3015–3023. method, MALDI tandem TOF mass spectrom- 9. Wang, D., Thompson, P., Cole, P. A., and eter provides a promising tool in analyzing Cotter, R. J. (2005) Structural analysis of a highly acetylated protein using a curved-field protein and peptide phosphorylation. reflectron mass spectrometer. Proteomics 5, 2288–2296. Acknowledgments 10. Håkansson, K., Chalmers, M. J., Quinn, J. P., The authors wish to thank Dr. Akhilesh McFarland, M. A., Hendrickson, C. L., and Pandey at the Johns Hopkins University for Marshall, A. G. (2003) Combined electron capture and infrared multiphoton dissociation for providing two commercial peptides. This multistage MS/MS in a fourier transform ion work was supported by a contract BAAHL- cyclotron resonance mass spectrometer. Anal. 02-04 from the National Heart Lung and Blood Chem. 75, 3256–3262.

Volume 2, 2006 ______Clinical Proteomics 09_Cotter 10/19/07 7:57 AM Page 144

144 ______Wang, Cole, and Cotter

11. Zubarev, R. A., Kelleher, N. L., and McLafferty, occur under matrix-assisted laser desorption F. W. (1998) Electron capture dissociation of ionization post-source decompositions and multiply charged protein cations. A non-ergodic matrix-assisted laser desorption collisionally process. J. Am. Chem. Soc. 120, 3265–3266. activated decompositions? J. Amer. Soc. Mass 12. Leymarie, N., Costello, C. E., O’Connor, P. B. Spectrom. 10, 217–223. (2003) Electron capture dissociation initiates a 18. Cheng, C., Pittenauer, E., and Gross, M. (1998) free radical reaction cascade. J. Am. Chem. Soc. Charge-remote fragmentations are energy- 125, 8949–8958. dependent processes. J. Amer. Soc. Mass Spec- 13. Stenballe, A., Jensen, O. N., Olsen, J. V., Hasel- trom. 9, 840–844. mann, K. F., and Zubarev, R. A. (2000) Electron 19. Kaufmann, R., Spengler, B., and Lutzenkirchen, capture dissociation of singly and multiply F. (1993) Mass spectrometric sequencing phosphorylated peptides. Rapid Commun. of linear peptides by product-ion analysis Mass Spectrom. 14, 1793–1800. in a reflectron time-of-flight mass spectrome- 14. Shi, S. D. -H., Hemling, M. E., Carr, S. A., ter using matrix-assisted laser desorption Horn, D. A., Lindh, I., and McLafferty, F. W. ionization. Rapid Commun. Mass Spectrom. 7, (2001) Phosphopeptide/phosphoprotein map- 902–910. ping by electron capture dissociation mass 20. Cotter, R. J., Gardner, B., Iltchenko, S., and spectrometry. Anal. Chem. 73, 19–22. English, R. D. (2004) Tandem time-of-flight 15. Cooper, H. J., Hakansson, K., and Marshall, A. G. mass spectrometry with a curved field reflec- (2005) The role of electron capture dissociation tron. Anal. Chem. 76, 1976–1981. in biomolecular analysis. Mass Spectrometry 21. Wang, D., Kalb, S. R., and Cotter, R. J. (2004) Reviews 24, 201–222. Improved procedures for N-terminal sulfona- 16. Taylor, G. K., Kim, Y. B., Forbes, A. J., Meng, F., tion of peptides for MALDI PSD peptide McCarthy, R., and Kelleher, N. L. (2003) Web sequencing. Rapid. Commun. Mass Spectrom. 18, and database software for identification of 96–102. intact proteins using “top down” mass spec- 22. Keough, T., Youngquist, R. S., and Lacey, M. P. trometry. Anal. Chem. 75, 4081–4086. (2003) Sulfonic acid derivatives for peptide 17. Rosario, M., Domingues, M., Marques, G. O. S., sequencing by MALDI MS. Anal. Chem. 75, et al. (1999) Do charge-remote fragmentations 157A–165A.

Clinical Proteomics ______Volume 2, 2006