Phosphoric Acid as a Matrix Additive for MALDI MS Analysis of

in the figure legends. A Voyager STR instrument (Applied ..... cantly enhanced in MALDI MS peptide mass maps and in peptide mass spectra obtained by M...
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Anal. Chem. 2004, 76, 5109-5117

Phosphoric Acid as a Matrix Additive for MALDI MS Analysis of Phosphopeptides and Phosphoproteins Sven Kjellstro 1 m and Ole Nørregaard Jensen*

Protein Research Group, Department of Biochemistry & Molecular Biology, University of Southern Denmark, DK-5230 Odense M, Denmark

Phosphopeptides are often detected with low efficiency by MALDI MS analysis of peptide mixtures. In an effort to improve the phosphopeptide ion response in MALDI MS, we investigated the effects of adding low concentrations of organic and inorganic acids during peptide sample preparation in 2,5-dihydroxybenzoic acid (2,5-DHB) matrix. Phosphoric acid in combination with 2,5-DHB matrix significantly enhanced phosphopeptide ion signals in MALDI mass spectra of crude peptide mixtures derived from the phosphorylated proteins r-casein and β-casein. The beneficial effects of adding up to 1% phosphoric acid to 2,5-DHB were also observed in LC-MALDI-MS analysis of tryptic phosphopeptides of B. subtilis PrkC phosphoprotein. Finally, the mass resolution of MALDI mass spectra of intact proteins was significantly improved by using phosphoric acid in 2,5-DHB matrix. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS)1,2 is a robust and sensitive analytical method in protein chemistry for the characterization of primary structure and in proteomics for the identification of proteins by peptide mass fingerprinting.3 During the past few years, the versatility of MALDI MS in protein chemistry and proteomics has been further extended by introduction of MALDI tandem mass spectrometers, including MALDI ion trap MS/MS,4,5 MALDI Q-TOF MS/MS,6 MALDI TOF-TOF MS/MS,7 and MALDI FT-ICR MS/MS.8 In contrast to these rapid developments in MALDI mass spectrometer instrumentation, the methods used for peptide and protein sample preparation have remained largely the same for the past decade. Sample preparation conditions used for peptide mass fingerprinting in MALDI MS usually include a 0.5-10 mg/mL * Corresponding author. Tel: +45 6550 2368. Fax: +45 6550 2467. URL: www.protein.sdu.dk. E-mail: [email protected]. (1) Karas, M.; Bachmann, D.; Bahr, U.; Hillenkamp, F. Int. J. Mass Spectrom. Ion Processes 1987, 53-68. (2) Karas, M.; Hillenkamp, F. Anal. Chem. 1988, 60, 2299-2301. (3) Jensen, O. N.; Larsen, M. R.; Roepstorff, P. Proteins: Struct. Genet. Suppl. 1998, 2, 74-89. (4) Qin, J.; Fenyo, D.; Zhao, Y. M.; Hall, W. W.; Chao, D. M.; Wilson, C. J.; Young, R. A.; Chait, B. T. Anal. Chem. 1997, 69, 3995-4001. (5) Krutchinsky, A. N.; Kalkum, M.; Chait, B. T. Anal. Chem. 2001, 73, 50665077. (6) Shevchenko, A.; Loboda, A.; Ens, W.; Standing, K. G. Anal. Chem. 2000, 72, 2132-2141. (7) Medzihradszky, K. F.; Campell, J. M.; Baldwin, M. A.; Falick, A. M.; Juhasz, P.; Vestal, M.; Burlingame, A. L. Anal. Chem. 2000, 72, 552-558. (8) Brock, A.; Horn, D. M.; Peters, E. C.; Shaw, C. M.; Ericsson, C.; Phung, Q. T.; Salomon, A. R. Anal. Chem. 2003, 75, 3419-3428. 10.1021/ac0400257 CCC: $27.50 Published on Web 08/06/2004

© 2004 American Chemical Society

solution of matrix, typically R-cyano-4-hydroxycinnamic acid (CHCA)9 or 2,5-dihydroxybenzoic acid (2,5-DHB)10 in a 50% organic solvent that also contains an organic acid, such as formic acid or trifluoroacetic acid. Studies aimed at improving sample preparation for MALDI MS analysis of peptides and proteins include, for example, investigations of solvent composition,11,12 analyte/matrix deposition strategies,13-18 influence of additives/ comatrixes,19,20 application of matrix mixtures,21 prestructured MALDI probes,22 and in situ liquid-liquid extraction for separation of hydrophilic and hydrophobic peptides.23 Determination of posttranslational modifications (PTMs) in proteins presents a number of challenges to mass spectrometry, particularly to sample preparation techniques.24 As an example, phosphorylated peptides are often more difficult to detect and analyze by MALDI MS than the corresponding unmodified species. Eliminating or reducing ion suppression effects and thereby improving the signal-to-background ratio for peptides in mass spectra has been the goal for a number of research groups who want to obtain fundamental insights into this effect25,26 or who want to apply MALDI mass spectrometry to study phosphorylated (9) Beavis, R. C.; Chaudhary, T.; Chait, B. T. Org. Mass Spectrom. 1992, 27, 156-158. (10) Strupat, K.; Karas, M.; Hillenkamp, F. Int. J. Mass Spectrom. 1991, 111, 89-102. (11) Cohen, S. L.; Chait, B. T. Anal. Chem. 1996, 68, 31-37. (12) Zhou, W.; Merrick, B. A.; Khaledi, M. G.; Tomer, K. B. J. Am. Soc. Mass Spectrom. 2000, 11, 273-282. (13) Xiang, F.; Beavis, R. C. Rapid Commun. Mass Spectrom. 1993, 8, 199-204. (14) Vorm, O.; Roepstorff, P.; Mann, M. Anal. Chem. 1994, 66, 3281-3287. (15) Kussmann, M.; Nordhoff, E.; Rahbeck-Nielsen, H.; Haebel, S.; Rossel-Larsen, M.; Jakobsen, L.; Gobom, J.; Migrorodskaya, E.; Kroll-Kristensen, A.; Palm, L.; Roepstorff, P. J. Mass Spectrom. 1997, 32, 593-601. (16) O ¨ nnerfjord, P.; Ekstro ¨m, S.; Bergquist, J.; Nilsson, J.; Laurell, T.; MarkoVarga, G. Rapid Commun. Mass Spectrom. 1999, 13, 315-322. (17) Hensel, R. R.; King, R. C.; Owens, K. G. Rapid Commun. Mass Spectrom. 1997, 11, 1785-1793. (18) Axelsson, J.; Hoberg, A.-M.; Waterson, C.; Myatt, P.; Shield, G. L.; Varney, J.; Haddleton, D. M.; Derrick, P. J. Rapid Commun. Mass Spectrom. 1997, 11, 209-213. (19) Gusev, A. I.; Wilkinson, W. R.; Proctor, A.; Hercules, D. M. Anal. Chem. 1995, 67, 1034-1041. (20) Billeci, T. M.; Stults, J. T. Anal. Chem. 1993, 65, 1709-1718. (21) Laugesen, S.; Roepstorff, P. J. Am Soc. Mass Spectrom. 2003, 14, 9921002. (22) Schuerenberg, M.; Luebbert, C.; Eickhoff, H.; Kalkum, M.; Lehrach, H.; Nordhoff, E. Anal. Chem. 2000, 72, 3436-3442. (23) Kjellstro ¨m, S.; Jensen, O. N. Anal. Chem. 2003, 75, 2362-2369. (24) Mann, M.; Jensen, O. N. Nat. Biotechnol. 2003, 415. (25) Knochenmuss, R.; Dubois, F.; Dale, M. J.; Zenobi, R. Rapid Commun. Mass Spectrom. 1996, 10, 871-877. (26) Amado, F. M. L.; Domingues, P.; Santana-Marques, M. G.; Ferrer-Correia, A. J.; Tomer, K. B. Rapid Commun. Mass Spectrom. 1997, 11, 1347-1352.

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proteins. At least five different approaches have been developed in order to improve the detection of phosphopeptides in MALDI MS: (i) chemical elimination and modification of phosphate group;27,28 (ii) selective enrichment of phosphopeptides using immobilized metal affinity chromatography (IMAC);29-32 (iii) use of additives/comatrixes such as ammonium salts to reduce alkali metal ion adduction;33 (iv) comparison of positive and negative mode;34,35 and (v) enzymatic removal of phosphate groups by treatment with alkaline phosphatase.36-38 The use of matrix additives or comatrixes to enhance phosphopeptide response in MALDI MS is a very appealing approach, since no chemical or enzymatic conversions are required nor are selective chromatographic methods necessary. The use of 2,5DHB with other substituted N.N-DHB derivatives for the enhanced detection of proteins was reported by Strupat et al.39 In our laboratory, we routinely use 2,5-DHB as a matrix for mass determination of phosphopeptides by MALDI MS. 2,5-DHB is known to be a “cool” matrix for MALDI; i.e., it leads to the formation of molecular ions with low internal energy, which remain intact during the mass analysis. This matrix produces rather heterogeneous and large crystalline deposits. In contrast, CHCA generates more homogeneous matrix/analyte deposits but is known as a “hot” matrix, which leads to significant decomposition of phosphopeptide ions during mass analysis, as observed by elimination of the phosphate group (-80 Da) or of phosphoric acid (-98 Da). In the present paper, we describe efforts to improve the performance of MALDI MS analysis for mass determination of phosphopeptides. Inspired by the observation that addition of strong acids to phosphopeptide samples leads to increased ion signal intensities for phosphopeptide ions in fast atom bombardment mass spectrometry,40 we set out to investigate whether organic and inorganic acids would enhance phosphopeptide response in MALDI MS using 2,5-DHB matrix. We found that the addition of 1% phosphoric acid to 2,5-DHB produced significantly improved phosphopeptide mass spectra. EXPERIMENTAL SECTION Proteins and Peptides. R- and β-casein were purchased from Sigma (St. Louis, MO). BSA for intact weight determination was obtained from Sigma. All aqueous solutions were prepared using (27) Meyer, H. E.; Hoffmann-Posorske, E.; Korte, H.; Heilmayer, L. M. J. FEBS Lett. 1986, 204, 61-66. (28) Oda, Y.; Nagasu, T.; Chait, B. T. Nat. Biotechnol. 2001, 19, 379-382. (29) Andersson, L. J. P. Anal. Biochem. 1986, 154, 250-254. (30) Stensballe, A.; Andersen, S.; Jensen, O. N. Proteomics 2001, 1, 207-222. (31) Neville, D. C. A.; Rozanas, C. R.; Price, E. M.; Gruis, D. B.; Verksam, A. S.; Townsend, R. R. Proteins Sci. 1997, 6, 2436-2445. (32) Posewitz, M. C.; Tempst, P. Anal. Chem. 1999, 71, 2883-2892. (33) Asara, J. M.; Allison, J. J. Am. Soc. Mass Spectrom. 1999, 10, 35-44. (34) Ma, Y.; Lu, Y.; Zeng, H.; Ron, D.; Mo, W.; Neubert, T. A. Rapid Commun. Mass Spectrom. 2001, 15, 1693-1700. (35) Janek, K.; Wenschuh, H.; Bienert, M.; Krause, E. Rapid Commun. Mass Spectrom. 2001, 15, 1593-1599. (36) Yip, T.-T.; Hutchens, T. W. FEBS Lett. 1992, 308, 149-153. (37) Zhang, X.; Herring, C. J.; Romano, P. R.; Szczepanowska, J.; Brzeska, H.; Hinnebusch, A. G.; Qin, J. Anal. Chem. 1998, 70, 2050-2059. (38) Larsen, M. R.; Sorensen, G. L.; Fey, S. J.; Larsen, P. M.; Roepstorff, P. Proteomics 2001, 1, 223-238. (39) Karas, M.; Ehring, H.; Nordhoff, E.; Stahl, B.; Strupat, K.; Hillenkamp, F.; Grehl, M.; Krebs, B. Org. Mass Spectrom. 1993, 28, 1476-1481. (40) Poulter, L.; Ang, S.-G.; Williams, D. H.; Cohen, P. Biochim. Biophys. Acta 1987, 929, 296-301.

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Milli-Q water filtered with a 0.2-µm membrane filter (Millipore, Bedford, MA). Proteolytic digestion of stock solutions of protein ( ∼2 pmol/µL) were performed using trypsin (modified/sequencing grade, Promega, Madison, WI) at an enzyme-to-substrate ratio of 1:100 at 37 °C for 4-18 h. The polypeptide mixtures generated were vacuum-dried and stored in the freezer until use. Sample Preparation for MALDI MS. 2,5-DHB was purchased from Aldrich Chemicals (Milwaukee, WI). Peptides were dissolved in 5% formic acid (FA), 0.1% trifluoroacetic acid (TFA), 2% phosphoric acid (PA), 0.1% n-octyl glucoside (all obtained from Sigma), or water to a final concentration of 0.05-0.5 pmol/µL. Subsequently, the sample (0.5 µL), was mixed in a 1:1 ratio with saturated matrix solution on the MALDI target using the dried droplet method.2 Saturated matrix solution was prepared by dissolving 2,5-DHB in 50% acetonitrile in water. LC-MALDI MS/MS on a Q-TOF Instrument. Peptide separation by capillary liquid chromatography (Ultimate, LC Packings, Amsterdam, The Netherlands) was accomplished by using an 8-cm analytical column (75-µm i.d., 360-µm o.d., Zorbax SB-C18 5 µm). The peptide mixtures were dissolved in buffer A (acetonitrile/water/formic acid; 2:97.9:0.1, v/v/v,), injected into the capillary LC system and separated by a linear gradient of 0-60% buffer B (acetonitrile/water/formic acid, 80:19.9:0.1, v/v/v) over 60 min. A robot system (Probot, LC Packings) was used for automatic deposition of the effluent from capillary liquid chromatography (flow rate ∼2 µL/min) onto the MALDI target in 1-min fractions. The matrix solution (2,5-DHB in 50% ACN/5% formic acid) was manually added to each position/fraction after the LC separation had finished. After initial screening of all fractions by MALDI MS, each analyte/matrix deposit was recrystallized using a solution of 1% phosphoric acid in 50% ACN and then new MALDI mass spectra were obtained. MALDI Mass Spectrometry. MALDI mass spectra of peptides were obtained using three different instruments as indicated in the figure legends. A Voyager STR instrument (Applied Biosystems, Framingham, MA) was used in either reflector or linear mode. The instrument was operated in the positive or negative ion delayed extraction mode. Ions were generated by irradiation of analyte/matrix deposits by a nitrogen laser at 337 nm and analyzed with an accelerating voltage of 25 kV in reflector mode and 20 kV in linear mode. MALDI mass spectra of peptides were also obtained using a Reflex IV MALDI reflector time-offlight mass spectrometer (Bruker-Daltonics, Bremen, Germany) equipped with a Scout-384 source. MALDI MS and MALDI MS/ MS spectra of peptide and peptide dimers were obtained using a MALDI Q-TOF instrument (Ultima HT, Waters/Micromass, Manchester, U.K.). Sample probes (MALDI targets) were made of polished stainless steel. All experiments were repeated several times with different amounts of sample (50-500 fmol) for peptide samples and 1-10 pmol for proteins, ensuring that the effects reported herein are reproducible. Collection of data for peptide fingerprints was performed in the same way independently on the instrument chosen. A series of different positions (5-8) within an analyte/matrix deposit were irradiated by the laser, and the mass spectrum obtained in each position was collected into a sum consisting of 400-800 mass spectra. When intact protein was investigated, only one represen-

Table 1. Overview of Phosphopeptides Investigated in This Studya peptide ID

AA no.

mass MH+

sequence

B&B index

R1 R2 R3 R4 β1 β2 P1 P2 P6 P5 P4 P3

121-134 58-73 119-134 74-94 33-48 1-25 208-221 280-291 150-183 150-183 150-183 150-183

1660.79 1927.69 1951.95 2720.91 2061.83 3122.27 1526.75 1606.68 3893.68 3813.72 3733.35 3653.78

YPQLEIVPN(pS)AEER DIG(pS)E(pS)TDQAMEDIK YKVPQLEIVPN(pS)AEER QMEA(pS)I(pS)(pS)(pS)EEIVPN(pS)VEQK FQ(pS)EEQQQTEDELQD RELEELNVPGEIVE(pS)L(pS)(pS)(pS)EESITR IPFDGE(pS)AVSIALK RFTIQEDEEM(pT)K VTDFGIATALSS[pT][pT]I[pT]H[pT]NSVLGSVHYLSPEQAR four phosphorylated sites three phosphorylated sites two phosphorylated sites one phosphorylated site

260 3960 -710 3770 6200 -120 -2450 1430 0 - 220 - 440 - 660

a Phosphopeptides from R-casein are denoted with Α1-Α4, and phosphopeptides from β-casein are denoted with β1 and β2. Phosphopeptides from PrkC are denoted P1-P6. B&B index calculated as described in Experimental Section. AA no. denotes amino acid residue number. MH+ denotes predicted m/z value.

tative position within an analyte/matrix deposit was used and a total of 100 laser shots were accumulated. Mass spectra obtained with the MALDI-Q-TOF instrument were investigated further using Masslynx software (Waters/ Micromass). All other mass spectra, data, and peptide sequence analysis were performed using m/z software (Proteometrics, Winnipeg, Canada) and GPMAW software (Lighthouse Data, Odense, Denmark), respectively. The Bull and Breese index was used to determine the hydrophobic/hydrophilic nature of peptides. The Bull and Breeze41 index for phosphopeptides was mimicked by changing phosphoserine or phosphothreonine residues to glutamic acid. RESULTS AND DISCUSSION Phosphopeptide Mapping of r-Casein by MALDI MS. During our recent work on the development of an in situ liquidliquid extraction method for MALDI MS,23 we became interested in the role of the organic acids that are typically added during sample preparation. We also noted that Poulter et al. in a study of phosphopeptide ion generation by fast atom bombardment mass spectrometry40 reported that the detection of hydrophilic analytes such as phosphopeptides could be improved by adding strong mineral acids to the FAB liquid sample matrix. Hence, in an effort to improve the sample preparation techniques for MALDI MS of phosphopeptide-containing samples we have investigated the effects of acids commonly used for MALDI sample preparation and several acids that are commonly used in liquid chromatography, i.e., heptafluorobutyric acid and phosphoric acid. A peptide mixture generated by tryptic digestion of R-casein was investigated by MALDI MS in both positive and negative ion reflector modes using 2,5-DHB matrix. Five different acids were used as matrix additives: acetic acid (AA), FA, PA, heptafluorobutyric acid (HFBA), and TFA. In some experiments we also included n-octyl glucoside in the peptide mixture since this additive is reported to facilitate solvation of peptides leading to improved peptide ion signal intensity and amino acid sequence coverage in peptide mass mapping experiments.42,43 2,5-DHB dissolved in 50% acetonitrile in water was used as the control matrix. The effect of acid addition to the matrix is demonstrated (41) Bull, H. B.; Breese, K. Arch. Biochem. Biophys. 1974, 161, 665-670.

by the following experiment. Aliquots of a tryptic peptide mixture of R-casein were redissolved in water, 0.1% TFA, and 2% PA, respectively. Each of these three samples was then mixed in a 1:1 ratio (v/v) with a saturated solution of 2,5-DHB matrix solution directly on the MALDI target. Phosphoric acid concentrations in the 0.01-10% range were investigated, and it was found that using a final concentration of 1% of phosphoric acid gave a high S/N intensity for the phosphopeptides without causing corrosion of the stainless steel target. Tryptic digestion of the 25.2-kDa R-casein phosphoprotein generates four phosphopeptides (R1, R2, R3, R4; see Table 1), in addition to numerous nonphosphorylated peptides. In the positive ion mode, the phosphopeptides R1 and R3 were reproducibly observed using any one of the four sample preparation conditions with addition of no acid, TFA, or PA (Figure 1A,C,E). The ion signal from the R2 phosphopeptide was rather low when water or TFA was used as a matrix additive (Figure 1A,C). However, phosphoric acid addition significantly enhanced this phosphopeptide ion signal, which now exceeded the intensity of the R1 phosphopeptide. (Figure 1E). In addition, the large, hydrophilic quadruply phosphorylated peptide R4 (m/z 2721, B&B 3770) from R-casein was only detected in the positive ion mode when PA was used as a matrix additive to 2,5-DHB (Figure 1E). Using either formic acid or heptafluorobutyric acid resulted in mass spectra similar to the ones obtained using water, 0.1% TFA, or n-octyl glucopyranoside, i.e., only three phosphopeptides R1,R2, and R3 could be observed whereas R4 escaped detection (data not shown). Furthermore, less sodiated peptide ions were detected when PA was used (Figure 1E). These results clearly indicated that PA addition to 2,5-DHB matrix enhances detection of R-casein phosphopeptides by MALDI MS. We next investigated whether the phosphopeptide ion detection could be further improved by switching to the negative ion mode in MALDI MS. Similar to the positive ion mode, the phosphopeptides R1, R2, and R3 were observed using any one of the four sample preparation conditions and negative ion MALDI MS (Figure 1, (42) Katayama, H.; Nagasu, T.; Oda, Y. Rapid Commun. Mass Spectrom. 2001, 15, 1416-1421. (43) Gobom, J.; Schuerenberg, M.; Mueller, M.; Theiss, D.; Lehrach, H.; Nordhoff, E. Anal. Chem. 2001, 73, 434-438.

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Figure 1. MALDI reflector TOF MS peptide mass fingerprints of 200 fmol of peptides obtained by tryptic digestion of R-casein. All samples were prepared using the dried droplet method using a saturated 2,5-DHB solution. Spectra were obtained in the positive ion mode (A, C, E) and the negative ion mode (B, D, F). Sample dissolved in water (A, B), 0.1% TFA (C, D), and 1% PA (E, F). Asterisks * indicates sodiated peptide ions. All mass spectra were obtained using the Voyager DE-STR MALDI TOF instrument.

panels B, D, F,). The R2 phosphopeptide ion exhibited a better signal-to-background ratio in the spectra obtained in the negative ion mode than in the positive ion mode. This was particularly obvious when using PA as a matrix additive to 2,5-DHB, which results in the R2 peptide being the base peak in the spectrum (Figure 1F). The large R4 phosphopeptide was detected in the negative ion mode when either TFA or PA was used as a matrix additive, but the signal intensity was significantly enhanced in the latter case (Figure 1 panels D and F). In general, we observed that in the negative ion mode the phosphopeptide ions were detected with a higher relative intensity as compared to other tryptic peptides. The absolute ion intensity was usually lower as compared to the positive ion mode, as also observed by others.34,35,44 The only notable exception to this observation is the spectrum obtained from 2,5-DHB matrix with addition of phosphoric acid, where the absolute phosphopeptide ion intensities observed in the negative ion mode were rather similar to those seen in the positive ion mode. Our conclusions from these experiments were that PA improved the detectability of phosphopeptides in both negative and positive ion modes. Since the negative ion MALDI MS mode in general produced fewer peptide ion signals in the low m/z region and is less sensitive for peptide mass mapping, we decided to use only positive ion MALDI MS in subsequent experiments.

Phosphopeptide Mapping of β-Casein. To investigate whether our observations were of a more general nature, we set out to apply PA as a matrix additive to 2,5-DHB for investigation of another model phosphoprotein, β-casein. A tryptic digest of β-casein is a commonly used sample for optimizing phosphopeptide analysis by MALDI MS30,33,36,38,45 as it contains two tryptic phosphopeptides. The β1 phosphopeptide observed at m/z 2062 contains one phosphoserine and is usually straightforward to detect by MALDI MS (Figure 2). The β2 phosphopeptide presents several challenges, as it is large and slightly hydrophobic (m/z 3122, B&B -120) and it contains four phosphoserine residues. Due to these features, the β2 phosphopeptide is often lost during sample preparation and it undergoes significant fragmentation during the MALDI MS analysis, as observed by abundant neutral loss of up to four PA moieties during reflector time-of-flight mass analysis. We have previously demonstrated that this particular phosphopeptide readily adsorbs to the surface of the stainless steel MALDI MS probe.23 Thus, the β2 ion signal is typically of low intensity or absent when subpicomole amounts of sample are used30 as also found in the present study where 100 fmol of peptide mixture was used (Figure 2, panels A and B). Nevertheless, addition of PA to the 2,5-DHB matrix solution resulted in a significant increase of the β2 phosphopeptide ion intensity (Figure 2, panel C).

(44) Wolfender, J. L.; Chu, F.; Ball, H.; Wolfender, F.; Fainzilber, M.; Baldwin, M. A.; Burlingame, A. L. J. Mass Spectrom. 1999, 34, 447-454.

(45) Liao, P.-C.; Leykam ,J.;Andrews, P. C.; Gage, D. A.; Allison, J. Anal. Biochem. 1994, 219, 9-20.

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Figure 2. Peptide fingerprint of a tryptic digestion of β-casein (100 fmol). All samples were prepared by the dried droplet method using a saturated 2,5-DHB solution. (A) Sample dissolved in water. (B) Sample dissolved in 0.1% TFA. (C) Sample dissolved in 1% PA. (D) Same analyte/matrix deposit as in (B) after recrystallization with 1% PA in 50% acetonitrile. The peptide ion signal at m/z 3129.7 corresponds to the peptide SLPQNIPPLTQTPVVWPPFLQPEVM*GVSK containing an oxidized methionine. All mass spectra were obtained in positive ion reflector mode using the Bruker Reflex MALDI TOF MS instrument.

We performed an additional set of experiments to ensure that the large β2 phosphopeptide was actually present in both the stock solution (after mixing with 0.1% TFA) and on the MALDI target used to generate the spectrum shown in Figure 2B. Recrystallization of the matrix/analyte deposit allows acquisition of additional mass spectra even after intensive laser ablation.46 Accordingly, we used 1% PA in 50% acetonitrile to redissolve the analyte/matrix deposit that originated from the 0.1% TFA solution, and then a new mass spectrum was obtained (Figure 2D). The β2 phosphopeptide was now easily detected as it generated an abundant molecular ion after recrystallization (Figure 2, compare panels B and D). This experiment proved that the β2 phosphopeptide was actually present in the TFA-containing analyte/matrix deposit; i.e., the absence of the β2 phosphopeptide molecular ion in Figure 2B was not due to sample loss via adsorption to vials or pipet tips. The experiment also shows that it is feasible to obtain (46) Page, J. S.; Sweedler, J. V. Anal. Chem. 2002, 74, 6200-6204.

consecutive mass spectra using 2,5-DHB matrix with various additives, i.e., first obtaining spectra with standard sample preparation conditions followed by recrystallization with a PAcontaining matrix to enhance phosphopeptide detection. We have found this feature particularly useful for phosphopeptide mapping by capillary liquid chromatography coupled off-line to MALDI MS/ MS as demonstrated below. Phosphopeptide Mapping by LC-MALDI-MS/MS. During recent years, we have established a number of analytical methods that enables sample preparation and detailed characterization of phosphoproteins by MALDI and ESI tandem mass spectrometry, including IMAC,30 LC-ESI-MS/MS,47-49 and MALDI MS/MS.50 We are currently investigating off-line coupling of capillary LC to MALDI MS and MALDI MS/MS using TOF-TOF, ion trap or Q-TOF instruments to establish whether such an approach provides any advantages over existing methods for phosphorylation site mapping of proteins. Off-line systems using automated fractionation are well suited for the interfacing of a liquid chromatographic separation into discrete matrix/analyte deposits51-54 or continuous tracks.55 Obviously, the sample preparation conditions chosen for converting the effluent from a liquid chromatographic separation into discrete matrix/analyte deposits will affect the results and information obtained from the MALDI MS experiment. In most cases, CHCA matrix is used for LCMALDI MS. This is due to the fact that analytes prepared by using CHCA matrix lead to homogeneous analyte/matrix deposits that are amendable to automated MALDI MS analysis.56 However, since CHCA is a relative hot matrix, more in-source fragmentation and postsource decay of fragile peptide ions occur. 2,5-DHB is a better matrix when posttranslational modifications (PTMs) such as phosphorylation are investigated. Hence, we studied whether addition of 2,5-DHB matrix with PA to LC fractions could enhance phosphopeptide ion signals obtained by MALDI MS analysis of 1-min LC fractions deposited on a stainless steel target. Matrix addition to HPLC eluent can be achieved in three principal ways: before, during, or after deposition of the eluting liquid onto the MALDI plate. In this initial study, we collected reversed-phase LC eluent into 1-min fractions directly on the MALDI probe and then added the matrix to the dried samples after the LC separation had completed. In this fashion, small analyte/matrix deposits were generated at all positions since the solvent composition of the LC gradient did not affect the viscosity of the matrix/analyte droplet. (47) Grønborg, M.; Kristiansen, T. Z.; Stensballe, A.; Andersen, J. S.; Ohara, O.; Mann, M.; Jensen, O. N.; Pandey, A. Mol. Cell. Proteomics 2002, 1, 517527. (48) Bykova, N. V.; Stensballe, A.; Egsgaard, H.; Jensen, O. N.; Moller, I. M. J. Biol. Chem. 2003, 278, 26021-26030. (49) Nuhse, T. S.; Stensballe, A.; Jensen, O. N.; Peck, S. C. Mol. Cell. Proteomics 2003, 2, 1234-1243. (50) Bennett, K. L.; Stensballe, A.; Podtelejnikov, A. V.; Moniatte, M.; Jensen, O. N. J. Mass Spectrom. 2002, 37, 179-190. (51) Bergman, A. C.; Bergman, T. J. Protein Chem. 1998, 67, 4197-4204. (52) Stevenson, T. I.; Loo, J. A. LC GC 1998, 16, 54-58. (53) Walker, K. L.; Chiu, R. W.; Moonig, C. A.; Wilkins, C. L. Anal. Chem. 1995, 67, 4197-4204. (54) Miliotis, T.; Kjellstro ¨m, S.; Nilsson, J.; Laurell, T.; Edholm, L. E.; MarkoVarga, G. J. Mass Spectrom. 2000, 35, 369-377. (55) Wall, D. B.; Berger, S. J.; Finch, J. W.; Cohen, S. A.; Chapman, R.; Drabble, D.; Brown, J.; Gostick, D. Electrophoresis 2002, 3193-3204. (56) Jensen, O. N.; Mortensen, P.; Vorm, O.; Mann, M. Anal. Chem. 1997, 69, 1706-1714.

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Figure 3. LC-MALDI MS spectra obtained from a tryptic digest of B. subtilis PrkC. The effluent from the capillary reversed-phase liquid chromatography separation was collected in one minute fractions on the MALDI plate. After completion of the LC run, a saturated solution of 2,5-DHB matrix (50% acetonitrile, 5% FA) was added to each position. Mass spectra from fraction at 21 min was obtained (A). The matrix/analyte deposit from fraction 21 was then redissolved in 1% PA in 50% ACN, and a new MALDI mass spectrum was obtained (B). Similarly, mass spectra from fraction at 30 min was obtained using standard conditions (C) and after addition of PA (D). Methionine oxidation of the m/z 2722.42 (EALNIMEQIVSAIAHAHQNQIVHR) caused by the recrystallization procedure is indicated in (D). All mass spectra obtained using the Waters/Micromass ULTIMA HT MALDIQ-TOF MS/MS instrument.

Using this setup, we analyzed a tryptic digest of the Bacillus subtilis PrkC phosphoprotein, which we have previously studied by electrospray ionization tandem mass spectrometry.57 A total of 60 fractions were collected and then analyzed by MALDI MS using standard conditions, i.e., 2,5-DHB matrix with addition of FA. As examples, the phosphopeptide P1 (m/z 1526.8) was detected by MALDI MS in fraction 21 and the phosphopeptide P3 (m/z 3653.78) was observed as a weak ion signal in fraction 30 (Figure 3, panels A and C). The latter spectrum also contained a weak signal at m/z 1628.8 that was tentatively assigned as a sodiated P2 phosphopeptide ion. Subsequently, these two analyte/ matrix deposits were recrystallized using addition of 1% PA and 50% ACN, as described in the previous sections. The MALDI MS spectrum obtained after recrystallization of fraction 21 showed a significantly enhanced ion signal from the P1 phosphopeptide, which was now the base peak in the spectrum (57) Madec, E.; Stensballe, A.; Kjellstro¨m, S.; Cladie`re, L.; Obuchowski, M.; Jensen, O. N.; Se´ror, S. J. J. Mol. Biol. 2003, 330 (3), 459-472.

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(Figure 3, panel B). MALDI MS analysis of fraction 30 generated a more intense P3 phosphopeptide ion and also enabled assignment of the P4 phosphopeptide ion, which corresponds to P3 but carries an additional phosphate moiety (Table 1). A low ion signal at m/z 1606.8 was now visible, which corresponded to the protonated P2 phosphopeptide ion, indicating that PA helps eliminate sodium adduction to phosphopeptides. These experiments and results further substantiate our general finding that PA in combination with 2,5-DHB matrix enhances phosphopeptide detection in MALDI MS. A more detailed investigation of the PrkC phosphoprotein by MALDI MS/MS will be presented elsewhere (Stensballe, A.; Jensen, O. N. Rapid Commun. Mass Spectrom. 2004, 18 (15), 1721-1730. Mass Determination of Intact Proteins. During our studies we observed that PA addition reduced the number of sodiumadducted peptide ions in MALDI mass spectra. Thus, one advantage of PA may be that it act as a “sodium sink” to capture residual sodium ions, thereby allowing more efficient generation of protonated peptide ions. To further investigate whether PA reduces the amount of sodium adduction of polypeptides, we used 2,5-DHB with addition of PA for mass determination of intact proteins, since alkali metal adduction often leads to “tailing” toward the high m/z side of protein ion signals. Several different proteins were investigated in the mass range from 5 (insulin) to 67 kDa (BSA). MALDI mass spectra obtained from R- and β-casein and BSA prepared in 2,5-DHB matrix containing low concentrations of either FA (control) or PA are depicted in Figure 4. Overall, less adduct formation by sodium ions was obtained when PA was used. As seen in Figure 4B, it was easier to distinguish between two different forms of R-casein when PA was used as compared to the control experiment (Figure 4A). The resolution in all mass spectra obtained with PA was significantly better than when FA was used (Figure 4). As an example, the resolution of the singly protonated β-casein ion signal varied between 120 and 150 full width at half-height (fwhm) in five individual mass spectra using PA (the resolution in Figure 4D is 130). When FA was used, the mass resolution varied between 40 and 80 (the mass resolution is 50 for the mass spectrum depicted in Figure 4C). In all mass spectra (Figure 4A-F), the highest peak was the singly charged protein ion. A notable difference is the relatively high abundance of doubly, triply, and (for larger proteins) quadruply protonated protein ions when using 2,5-DHB with PA. The observed increase in mass resolution may be due to reduced adduct formation but also due to a reduced internal energy of the ions generated from the PA-doped 2,5-DHB crystals. We have performed some preliminary experiments to further investigate these observations, as outlined in the following sections. Effects of Phosphoric Acid on Sample Preparation and Ion Formation in MALDI MS. During this study, we observed that the β1 and β2 phosphopeptides derived from β-casein generated doubly charged ions only when PA was used as matrix additive. This was not observed when using the same amount of sample (0.5 pmol of peptide mixture loaded on target) and 2,5DHB in combination with other acids or water. In addition, we observed a strong tendency of the β1 and β2 phosphopeptides to generate singly charged phosphopeptide homodimer and heterodimer ions, [β1β1 + H]+, [β2β2 + H]+, and [β1β2 + H]+, which were observed in both linear and reflector TOF modes,

Figure 4. Improved resolution of protein mass spectra. MALDI mass spectra of intact R-casein (2 pmol, A and B), β-casein (2 pmol, C and D), and BSA (1 pmol, E and F). Spectra in (A-C) were obtained using 2,5-DHB in 5% FA/50% acetonitrile and spectra in (B, D, F) were obtained using 2,5-DHB in 1% PA/50% acetonitrile. All mass spectra obtained in positive ion linear mode using the Applied Biosystems Voyager STR instrument. Each mass spectrum (100 laser shots) was obtained from a single position within the sample deposit.

but only after recrystallization using 2,5-DHB with PA as an additive (Figure 5). Only phosphopeptides and no unphosphorylated peptides produced such abundant dimer ions. We could confirm the identity of the m/z 4122.7 ion as [β1β1 + H]+ by MALDI Q-TOF MS/MS using a higher amount of sample (Figure 6). Due to the m/z 4200 upper limit for ion selection by the quadrupole ion filter in the Q-TOF MS/MS instrument, we were not able to investigate larger dimer ions. Generation of doubly protonated peptide ions and peptide dimer ions in MALDI MS is usually an indication of sample “overloading” during sample preparation or application of excessive laser fluence for irradiation of the sample deposit. Neither explanation applied in this case, as only 0.5 pmol of sample was used and laser energy slightly over threshold was applied. Indeed, using higher laser fluence resulted in a reduced signal intensity of these phosphopeptide dimer ions (data not shown). The observation of singly charged phosphopeptide dimer ions, singly charged phosphopeptide ions, and doubly charged phosphopeptide ions in the same mass spectrum suggests that PA improves the overall ionization efficiency, increases ion stability, or both. This observation has to be further investigated, but it is likely associated with the mechanism of cocrystallization of analyte with matrix molecules to form deposits amendable to MALDI MS. Thus, the sensitivity gain of phosphopeptide analysis by MALDI MS using PA as an additive to 2,5-DHB matrix may be explained by considering the inclusion mechanism for incorporation of analyte molecules into the crystalline matrix/analyte deposit. The

fact that recrystalization experiments leads to improved phosphopeptide ion signals supports this idea. It has been reported that solvent molecules are incorporated in the analyte/matrix lattice58 and that the analytes were incorporated as charged species in the host matrix crystals.59 Hence, we speculate that the phosphopeptides and proteins were incorporated in the matrix lattice with higher charged states when PA was used, as compared to when other acids or water was used. The PA solution will reach a very high ionic strength during the evaporation of solvent, thereby forcing protonated phosphopeptides to be incorporated into the growing crystalline matrix/analyte deposit. The presence of doubly charged ions and molecular cluster ions (homo- and heterodimers) of phosphopeptides but not of unphosphorylated peptides may be interpreted as being due to more efficient incorporation of charged phosphopeptides into the crystalline analyte/matrix deposit during evaporation of the solvent, leading to a high concentration of (multiply) charged phosphopeptides and phosphopeptide clusters in the matrix crystals. In this context, the acidity of the acids seems not to play a role, as some of the other acids we have investigated are significantly more acidic (TFA, HFBA) or more basic (acetic acid, water) than PA. Instead, we hypothesize that “salting out” of (58) Horneffer, V.; Dreisewerd, K.; Ludemann, H. C.; Hillenkamp, F.; Lage, M.; Strupat, K. Int. J. Mass Spectrom. 1999, 187, 859-870. (59) Kruger, R.; Pfenninger, A.; Fournier, I.; Gluckmann, M.; Karas, M. Anal. Chem. 2001, 73, 5812-5821.

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Figure 5. Tryptic peptide fingerprint of β-casein (500 fmol deposit) revealing dimer ion formation. All samples were prepared using the dried droplet method using a saturated solution of 2,5-DHB. (A) Sample dissolved in water. (B) Sample dissolved in 5% FA. (C) Sample dissolved in 2% PA. Peptide ion at m/z 3113.7 is SLPQNIPPLTQTPVVWPPFLQPEVMGVSK (methionine oxidation, m/z 3129.7). Two regions, m/z 31003150 and 4000-7000, marked with gray boxes are magnified in each mass spectrum. Mass spectrum in the linear mode depicted between m/z 4000 and 7000 clearly shows the homo- and heterodimers. Note that the singly charged dimer ions, m/z 4122 [β1β1]+, 5183 [β1β2]+, and m/z 6122 [β2β2]+ were only present when PA was used. All mass spectra were obtained in positive ion reflector and linear modes as indicated, using the Applied Biosystems Voyager STR instrument.

Figure 6. MALDI Q-TOF tandem mass spectra of the β-casein b1 phosphopeptide monomer and dimer ion species. (A) Singly protonated phosphopeptide dimer ion [β1β1 + H]+ at m/z 4124. (B) Singly protonated phosphopeptide ion [β1 + H]+ at m/z 2061.8.

phosphopeptides by the PA anion is leading to more efficient incorporation of such phosphopeptides into the growing 2,5-DHB 5116

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crystals. We also note that according to the Hofmeister series PA is one of the most potent anions for “salting out” polypeptides.

CONCLUSION We have demonstrated that PA serves as a useful matrix additive for MALDI MS analysis of phosphopeptides when using 2,5-DHB as the matrix. Phosphopeptide ion signals were significantly enhanced in MALDI MS peptide mass maps and in peptide mass spectra obtained by MALDI MS analysis of LC fractions. ACKNOWLEDGMENT Drs. Per Ha¨gglund, Sabrina Laugesen, Allan Stensballe, and Thomas J. D. Jørgensen are acknowledged for helpful discussions.

The Danish Natural Sciences Research Council and Danish Biotechnology Instrument Center is acknowledged for support. S.K. was financed by an EU research grant.

Received for review February 13, 2004. Accepted June 9, 2004. AC0400257

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