Atmospheric Pressure MALDI-Fourier Transform Mass Spectrometry

Anal. Chem. 2004, 76, 3930-3934

Atmospheric Pressure MALDI-Fourier Transform Mass Spectrometry Katherine A. Kellersberger,† Phillip V. Tan,‡ Victor V. Laiko,‡ Vladimir M. Doroshenko,‡ and Daniele Fabris*,†

Department of Chemistry/Biochemistry, University of Maryland, Baltimore County, Baltimore, Maryland 21250, and MassTech Inc., Columbia, Maryland 21046

The coupling of atmospheric pressure matrix-assisted laser desorption/ionization (AP MALDI) with Fourier transform mass spectrometry (FTMS) is described, and its significance for the high-resolution analysis of complex peptide mixtures is demonstrated. High kinetic energy and extensive metastable decay characteristic of ions generated by vacuum MALDI have been known to constitute a possible obstacle to high-resolution analysis by FTMS. Since the initial coupling of laser desorption techniques with FTMS was realized two decades ago, several different solutions have been proposed to control the energy of the ions and fulfill the promise of high sensitivity and high resolution offered by this analytical method. Initial results obtained on quadrupole time-offlight and ion trap analyzers have shown that ions generated by MALDI at atmospheric pressure are intrinsically less energetic than those provided by vacuum MALDI. Our report indicates that this characteristic is particularly beneficial for FTMS applications in which a sharp reduction of metastable decay can make larger ion currents available for detection and possible tandem experiments. In our hands, AP MALDI-FTMS has enabled the analysis of complex peptide mixtures with resolution and accuracy comparable to those obtained by analogous electrospray ionization-FTMS experiments, with no evidence of either metastable decomposition or significant formation of matrix adducts. Analysis of a trypsin digest of bovine serum albumin provided signal-to-noise ratios and limits of detection similar to those obtained by ion trap analyzers, but with unmatched resolution and accuracy. AP MALDI has been shown to provide stable precursor ions in amounts that allowed for informative tandem experiments. Finally, the potential of AP MALDI-FTMS for the high-resolution screening of complex mixtures was demonstrated by the analysis of isobaric peptides differing in mass by less than 0.04 Da. The possibility of combining laser-based ionization techniques with Fourier transform mass spectrometry (FTMS)1,2 and the potential for high-resolution analysis of polar, nonvolatile, and * Corresponding author. Tel.: (410) 455-3053. Fax: (410) 455-2608. E-mail: [email protected]. † University of Maryland. ‡ MassTech Inc. (1) Comisarow, M. B.; Marshall, A. G. Chem. Phys. Lett. 1974a, 25, 282-283.

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thermally labile compounds were realized two decades ago with the introduction of laser desorption-FTMS.3-5 The subsequent development of matrix-assisted laser desorption/ionization (MALDI)6,7 brought this potential to fruition for the investigation of progressively larger biomolecules and polymers.8-12 At the time, the leading instrumental approach involved focusing a laser beam onto a sample probe located in the immediate proximity of one of the trapping plates of the cell, thus admitting ions and neutral products generated by the laser desorption event directly into the cell.12 Unfortunately, the high kinetic energy13 and extensive metastable decay14,15 of ions generated by MALDI made trapping and FTMS detection quite difficult and contributed to unexpectedly poor instrumental performance.12 Different solutions were proposed to solve this problem and fulfill the promise of high resolution and sensitivity by MALDI-FTMS. These included alternative trapping and axialization schemes, addition of sugar comatrixes, and application of pulsed gas to achieve collisional relaxation.10,11,16-21 Instrument design modifications included the addition of a “waiting room” chamber to cool ions before injection (2) Marshall, A. G.; Hendrickson, C. L.; Jackson, G. S. Mass Spectrom. Rev. 1998, 17, 1-35. (3) Cody, R. B.; Burnier, R. C.; Reents, W. D. J.; Carlin, T. J.; McCrery, D. A.; Lengal, R. K.; Freiser, B. S. Int. J. Mass Spectrom. Ion Phys. 1980, 33, 3743. (4) McCrery, D. A.; Ledford, E. B. J.; Gross, M. L. Anal. Chem. 1982, 54, 14351437. (5) Wilkins, C. L.; Weil, D. A.; Yang, C. L. C.; Ijames, C. F. Anal. Chem. 1985, 57, 520-524. (6) Tanaka, K.; Ido, H.; Yoshida, Y.; Yoshida, T. Proceedings of the Second JapanChina Joint Symposium on Mass Spectrometry; Bando Press: Osaka, Japan, 1987; pp 185-187. (7) Karas, M.; Hillenkamp, F. Anal. Chem. 1988, 60, 2299-2301. (8) Hettich, R. L.; Buchanan, M. V. J. Am. Soc. Mass Spectrom. 1991, 2, 2228. (9) Hettich, R.; Buchanan, M. J. Am. Soc. Mass Spectrom. 1991, 2, 402-412. (10) Castoro, J. A.; Ko¨ster, C.; Wilkins, C. L. Rapid Commun. Mass Spectrom. 1992, 6, 239. (11) Koster, C.; Castoro, J. A.; Wilkins, C. L. J. Am. Chem. Soc, 1992, 114, 75727574. (12) Buchanan, M. V.; Hettich, R. L. Anal. Chem. 1993, 65, 245A-259A. (13) Pan, Y.; Cotter, R. J. Org. Mass Spectrom. 1992, 27, 3-8. (14) Spengler, B.; Kirsch, D.; Kaufmann, R. Rapid Commun. Mass Spectrom. 1991, 5, 198-202. (15) Cotter, R. J. Anal. Chem. 1992, 64, 1027A. (16) Rempel, D. L.; Gross, M. L. J. Am. Soc. Mass Spectrom. 1992, 3, 590. (17) Schweikhard, L.; Guan, S.; Marshall, A. G. Int. J. Mass Spectrom. Ion Processes 1992, 120, 71-83. (18) Guan, S.; Whal, M. C.; Wood, T. D.; Marshall, A. G. Anal. Chem. 1993, 65, 1753. (19) Pastor, S. J.; Castoro, J. A.; Wilkins, C. L. Anal. Chem. 1995, 67, 379-384. (20) Yao, J.; Dey, M.; Pastor, S. J.; Wilkins, C. L. Anal. Chem. 1995, 67, 36383642. 10.1021/ac0498415 CCC: $27.50

© 2004 American Chemical Society Published on Web 05/28/2004

into the actual analyzer cell22 or the insertion of a wire guide between the trapping plates.23 An important breakthrough came with the introduction of MALDI external sources24-27 on the wings of the development of similar apparatus for interfacing FTMS to liquid secondary ionization mass spectrometry28-30 and electrospray ionization.31-33 According to this design, ions are produced by MALDI in a region located outside the magnet, extracted from the source by an electric field, transported through the fringing fields of the magnet by an ion guide formed by multipolar or electrostatic elements, and finally injected into the analyzer cell located within the bore of the superconducting magnet. In this way, the processes of ionization, trapping, and analysis can be independently optimized for best performance. On one hand, several stages of differential pumping can better serve to keep the analyzer pressure to optimal levels in the wake of frequent sample changes. On the other hand, the time scale of a typical experiment in FTMS with an external source may stretch from milliseconds to seconds, as compared to microseconds for analogous time-of-flight (TOF) experiments,34 thus extending significantly the time available for possible metastable decomposition of MALDI-generated ions.35-37 Metastable processes are known to cause significant deterioration of the overall detection sensitivity for decaying analytes, affect the ability of observing labile posttranslational modifications in peptides and nucleic acids, and compete unfavorably with other activation methods in tandem techniques. Collisional cooling in the source region was proposed as an effective way to reduce the kinetic and vibrational energy of ions generated by MALDI for TOF detection38 in analogy with earlier methods employing pulsed gas in MALDI-FTMS instruments with internal sources,17-21 Using this approach, the plume of charged and neutral species is allowed to expand into a quadrupole, which (21) Pasa Tolic, L.; Huang, S.; Guan, S.; Kim, H. S.; Marshall, A. G. J. Mass Spectrom. 1995, 30, 825. (22) Solouki, T.; Russell, D. H. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 57015704. (23) Solouki, T.; Gillig, K. J.; Russell, D. H. Anal. Chem. 1994, 66, 1583-1587. (24) McIver, R. T. J.; Li, Y.; Hunter, R. L. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 4801-4805. (25) Li, Y.; McIver, R. T. J.; Hunter, R. L. Anal. Chem. 1994, 66, 2077-2083. (26) Wu, J.; Fannin, S. T.; Franklin, M. A.; Molinski, T. F.; Lebrilla, C. B. Anal. Chem. 1995, 67, 3788-3792. (27) Heeren, R. M.; Boon, J. J. Int. J. Mass Spectrom. Ion Processes 1996, 157/ 158, 391-403. (28) McIver, R. T. J.; Hunter, R. L.; Bowers, W. D. Int. J. Mass Spectrom. Ion Processes 1985, 64, 67-77. (29) Hunt, D. F.; Shabanowitz, J.; Yates, J. R.; Zhu, N. Z.; Russell, D. H.; Castro, M. E. Proc. Natl. Acad. Sci. U.S.A. 1987, 84, 60-63. (30) Lebrilla, C. B.; Amster, I. J.; McIver, R. T. J. Int. J. Mass Spectrom. Ion Processes 1989, 87, R7-R13. (31) Henry, K. D.; Williams, E. R.; Wang, B.-H.; McLafferty, F. W.; Shabanowitz, J.; Hunt, D. F. Proc. Natl. Acad. Sci. U.S.A. 1989, 86, 9075-9078. (32) Henry, K. D.; Quinn, J. P.; McLafferty, F. W. J. Am. Chem. Soc, 1991, 113, 5447-5449. (33) Winger, B. E.; Hofstadler, S. A.; Bruce, J. E.; Udseth, H. R.; Smith, R. D. J. Am. Soc. Mass Spectrom. 1993, 4, 566-577. (34) Cotter, R. J. Time-of-Flight Mass Spectrometry. Instrumentation and applications in biological research; American Chemical Society: Washington, DC, 1997. (35) Cancilla, M. T.; Penn, S. G.; Carroll, J. A.; Lebrilla, C. B. J. Am. Chem. Soc, 1996, 118, 6736-6745. (36) Penn, S. G.; Cancilla, M. T.; Lebrilla, C. B. Anal. Chem. 1996, 68, 23312339. (37) Ho, Y.-P.; Fenselau, C. C. J. Mass Spectrom. 2000, 35, 183-188. (38) Krutchinsky, A. N.; Loboda, A. V.; Spicer, V. L.; Dworschak, R.; Ens, W.; Standing, K. G. Rapid Commun. Mass Spectrom. 1998, 12, 508-518.

is kept at ∼0.1 mbar by a constant flow of nitrogen through a leak valve. Ions are decelerated and cooled by collisions before undergoing extraction into an orthogonal TOF analyzer.38 A similar strategy has been successfully demonstrated in MALDI-FTMS instruments39-41 equipped with external sources that were modified to include an rf-only multipole ion reservoir.42,43 In these experiments, MALDI is carried out at the entrance of the ion reservoir, while gas can be pulsed to raise the source pressure to a maximum of ∼10 mbar. Collisionally dampened ions are subsequently injected in a timely fashion through the ion guide and into the analyzer cell, showing a greatly reduced incidence of metastable decomposition.40 In this report, we test the possibility of utilizing low-energy ions produced at substantially higher pressure for FTMS analysis. This approach is based on the implementation of the recently developed atmospheric pressure (AP) MALDI,44,45 which involves focusing a laser beam onto a sample plate located in the immediate proximity of the inlet of an electrospray ionization (ESI) interface.46 In much the same way as electrosprayed droplets are sampled at atmospheric pressure and ions undergo desolvation through collisions in the interface, a portion of the MALDI plume is drawn into the inlet of the ESI source, where ions are subjected to collisional relaxation. The evidence provided by extensive testing in both quadrupole time-of-flight (Q-TOF)45,47,48 and ion trap mass spectrometers49-53 indicates that ions generated at atmospheric pressure are significantly less energetic than those provided by traditional vacuum MALDI and do not undergo the same extent of metastable decay. The significance of making these ions available for high-resolution analysis is explored in this work by coupling an AP MALDI source with an FTMS analyzer equipped with a commercial atmospheric pressure ionization (API) interface. EXPERIMENTAL SECTION Angiotensin I, angiotensin II, fibrinopeptide A, substance P, melittin, bradykinin, and Leu-enkephalin-Lys (YGGFLK) were obtained from Sigma Chemical Co. (St. Louis, MO) and used without further purification. Bovine serum albumin (BSA) tryptic (39) Baykut, G.; Jertz, R.; Witt, M. Rapid Commun. Mass Spectrom. 2000, 14, 1238-1247. (40) O’Connor, P. B.; Costello, C. E. Rapid Commun. Mass Spectrom. 2001, 15, 1862-1868. (41) O’Connor, P. B.; Budnik, B. A.; Ivleva, V. B.; Kaur, P.; Moyer, S. C.; Pittman, J. L.; Costello, C. A. J. Am. Soc. Mass Spectrom. 2004, 15, 128-132. (42) Senko, M. W.; Hendrickson, C. L.; Emmett, M. R.; Stone, D.-H.; Marshall, A. G. J. Am. Soc. Mass Spectrom. 1997, 8, 970-978. (43) Sannes-Lowery, K. A.; Griffey, R. H.; Kruppa, G. H.; Speir, J. P.; Hofstadler, S. A. Rapid Commun. Mass Spectrom. 1998, 12, 1957-1961. (44) Laiko, V. V.; Burlingame, A. L. U.S. Patent 5965884, 1999. (45) Laiko, V. V.; Baldwin, M. A.; Burlingame, A. L. Anal. Chem. 2000, 72, 652657. (46) Yamashita, M.; Fenn, J. B. J. Phys. Chem. 1984, 88, 4671-4675. (47) Harvey, D. J.; Bateman, R. H.; Bordoli, R. S.; Tyldesley, R. Rapid Commun. Mass Spectrom. 2000, 14, 2135-2142. (48) McLean, J. A.; Russell, W. K.; Russell, D. H. Anal. Chem. 2003, 75, 648654. (49) Laiko, V. V.; Moyer, S. C.; Cotter, R. J. Anal. Chem. 2000, 72, 5239-5243. (50) Moyer, S. C.; Cotter, R. J.; Woods, A. S. J. Am. Soc. Mass Spectrom. 2001, 13, 274-283. (51) Laiko, V. V.; Taranenko, N. I.; Berkout, V. D.; Musselman, B. D.; Doroshenko, V. M. Rapid Commun. Mass Spectrom. 2002, 16, 1737-1742. (52) Galicia, M. C.; Vertes, A.; Callahan, J. H. Anal. Chem. 2002, 74, 18911895. (53) Miller, C. A.; Yi, D.; Perkins, P. D. Rapid Commun. Mass Spectrom. 2003, 17, 860-868.

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digest was obtained from Michrom BioResources (Auburn, CA) and used as received with 2.0% acetonitrile and 0.1% trifluoroacetic acid to make up stock solutions. The custom peptide Leuenkephalin-Gln (YGGFLQ) was purchased from Bio-Synthesis, Inc. (Houston, TX) and used without further purification. The matrix R-cyano-4-hydroxycinnamic acid was obtained from Fluka (St. Louis, MO). In general, analytes were dissolved and diluted in HPLC-grade water to prepare solutions of appropriate concentration. An aliquot of sample solution, typically 0.2-1.0 µL, was deposited onto the target plate and added with matrix in the appropriate volume (typically 1.0-1.8 µL) to make a 2-µL spot. After gentle mixing, the spot was allowed to dry under atmospheric conditions. The MassTech Inc. (Columbia, MD) AP MALDI apparatus has been described previously54,55 and includes a control unit and ion source flange. For this work, the commercially available AP/ MALDI model 221 was used. Photons are produced by a nitrogen laser (337 nm) contained in the control unit and are transmitted to the ion source via an optical fiber cable (400-µm diameter). In the ion source, they are focused by a quartz lens and reflected by a mirror to impinge the sample target with a 45° angle. The sample plate is held magnetically onto an X,Y stage, which can move in 0.0-0.5-mm steps under computer control. This enables the user to manually change the position of the spot exposed to the laser beam. Alternatively, the position of the sample plate can be automatically controlled by Target software (MassTech, Inc.) to move in a spiral pattern from the center of the sample spot. The AP MALDI source was coupled with a Bruker (Billerica, MA) Apex III FTMS, equipped with a 7.0-T actively shielded magnet and Apollo electrospray interface, which had been modified to include a heated desolvation capillary. Such coupling required the replacement of the ESI flange with the AP MALDI flange, which is readily accomplished without breaking source vacuum. The high-voltage supply for the ESI needle was also rewired to the MALDI sample plate. During typical operation, the plate was held at a high-voltage potential (2.5-3.5 kV) relative to the inlet of the desolvation capillary to facilitate the sampling of the MALDI plume into the ESI interface. No drying gas was necessary, and the capillary temperature (TC) was varied in the 150-240 °C range according to the analyte. Each spectrum consisted of an average of 5-25 scans acquired in positive ion mode with a total accumulation time varying from 12.5 to 300 s, during which the laser was continuously fired with a 10-Hz repetition rate. The duration of each individual scan was determined in part by the time allowed for ions to accumulate in the external hexapole located immediately after the second skimmer in the electrospray source.43 For tandem experiments, ions of interest were isolated in the cell using correlated rf sweeps56 followed by activation through sustained off-resonance irradiation (SORI)57 against a background of argon gas. Typical frequency offsets were 600-1500 Hz below the frequency of the (54) Moyer, S. C.; Marzilli, L. A.; Woods, A. S.; Laiko, V. V.; Doroshenko, V. M.; Cotter, R. J. Int. J. Mass Spectrom. 2003, 226, 133-150. (55) Doroshenko, V. M.; Laiko, V. V.; Taranenko, N. I.; Berkout, V. D.; Lee, H. S. Int. J. Mass Spectrom. 2002, 221, 39-58. (56) de Koning, L. J.; Nibbering, N. M. M.; van Orden, S. L.; Laukien, F. H. Int. J. Mass Spectrom. Ion Processes 1997, 165/166, 209-219. (57) Gauthier, J. W.; Trautman, T. R.; Jacobson, D. B. Anal. Chim. Acta 1991, 246, 211-225.

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Figure 1. AP MALDI-FTMS analysis of a peptide mixture containing angiotensin I, angiotensin II, bradykinin, and fibrinopeptide A. The matrix was R-cyano-4-hydroxycinnamic acid (CHCA). Typical resolution was ∼100 000 M/∆Mfwhm, while accuracy was ∼2 ppm.

precursor ion. Mass assignments were based on a three-point external calibration obtained from standard peptides. RESULTS AND DISCUSSION Coupling AP MALDI with an FTMS instrument equipped with an external atmospheric pressure ionization interface enabled the high-resolution analysis of peptides present in complex mixtures (Figure 1) with resolution and accuracy comparable to those achieved in analogous ESI-FTMS experiments on the same analyzer (∼100 000 M/∆Mfwhm in broad-band mode and ∼2 ppm, respectively). The number and intensity of peaks arising from matrix debris, clusters, and other species, which are frequently observed in the low-mass range of vacuum MALDI spectra,8,9,12 were strikingly low. Pseudomolecular ions corresponding to protonated peptides were detected intact without the frequent loss of H2O or NH3 observed in a vacuum MALDI37 and with no evidence of metastable backbone fragmentation. This favorable feature can be credited not only to the “gentle” character of highpressure38-41 and AP MALDI,45,49,50 but also to thermalization processes occurring in the external rf hexapole reservoir,42,43 where ions were accumulated and stored for extensive periods of time (typically 0.5-3 s) before being transferred to the FTMS cell for analysis. No matrix-analyte adducts were detected in this peptide mixture, but abundant adducts were observed for larger analytes, as previously observed in AP MALDI experiments on a Q-TOF instrument.45 For example, matrix-analyte clusters were the only species detected for melittin (2845.761 Da, monoisotopic mass calculated from sequence) under conditions including a 150 °C capillary temperature (TC) and a 250-V difference in potential between the exit of the capillary and the first skimmer (∆VCS) (Figure 2a). Raising these parameters closer to the hardware limits (TC ) 240 °C and ∆VCS ) 280 V) improved the declustering process and allowed for the detection of the desired analyte, but did not completely eliminate the presence of matrix adducts (Figure 2b). It should be pointed out that a TC of 120 °C and a ∆VCS of 190 V are normally employed for the ESI analysis of analogous peptides with the same interface. The extreme settings,

Figure 2. AP MALDI-FTMS analysis of melittin obtained under (a) typical interface conditions (TC ) 150 °C and ∆VCS ) 250 V) and (b) harsher desolvation parameters (TC ) 240 °C and ∆VCS ) 280 V). *, FTMS alias.

which yielded incomplete declustering of AP MALDI ions, would have likely induced extensive in-source dissociation of the corresponding multiply charged ions generated by ESI, perhaps due to the greater kinetic energy imparted to such ions by an increased ∆VCS. One of the limitations of external source MALDI-FTMS has been the ability to generate ions on a time scale compatible with the duty cycle of the instrument, while preserving the number of pseudomolecular ions available for tandem analysis. In fact, extensive metastable decomposition has often been credited for causing significant depletion of the ion population of interest, making it difficult to obtain the signal necessary for collisioninduced dissociation (CID) or slow-heating experiments.37 It is not uncommon for precursor ions available in sufficient number for isolation in a tandem experiment to undergo extensive fragmentation prior to excitation due to their excessive internal energy. Precursor ions obtained by AP MALDI were found to survive intact the isolation step preceding excitation and collisional activation by SORI-CID. For example, the isolation of substance P provided a clean spectrum, with no evidence of metastable decomposition after being trapped in the cell for up to 5 s (Figure 3a). When subjected to collisional activation, the selected ion produced the abundant sequence ions58 typical of SORI-CID experiments (Figure 3b). Detection limits were found to be comparable to those determined on ion trap mass spectrometers under similar conditions.49,50,53 The analysis of 10 fmol of BSA digest (Figure 4a) allowed the observation of peptides covering 25% of the total protein sequence. Decreasing the amount of digest deposited on the target plate to 1 fmol reduced the coverage to 14%, and only the more intense species were observed with reasonable signalto-noise (S/N) ratios (Figure 4b). Greater S/N ratios were obtained by increasing the time allowed for ion accumulation in the external hexapole and, consequently, pooling together the ions desorbed by a larger number of laser shots before injecting them all at once into the ion guide for transfer to the FTMS cell. Alternatively, S/N ratios were improved by increasing the overall number of ions sampled from the MALDI plume into the inlet of the atmospheric pressure interface using a recently developed (58) Biemann, K. Annu. Rev. Biochem. 1992, 61, 977-1010.

Figure 3. Tandem mass spectrum of singly charged substance P desorbed by AP MALDI. The isolation spectrum (a) showed no visible metastable decomposition even after extended periods of time. The product ion spectrum (b) shows characteristic sequence ion series. All fragment ions are singly charged.

Figure 4. AP MALDI-FTMS analysis with the application of pulsed dynamic focusing (PDF) of (a) 10 and (b) 1 fmol of a tryptic digest of BSA. The insets show the difference between signal-to-noise ratios obtained with (left) and without (right) the application of PDF. *, FTMS alias.

pulsed dynamic focusing (PDF) interface.59 Briefly, standard AP MALDI analyses employ a continuous electric field to extract ions into the mass analyzer; however, many are discharged or lost due to collisions with the walls and tip of the desolvation capillary. By removing the electric field for a desired time interval immediately following the laser desorption event, ions are entrained by the gas flow into the entrance slit or desolvation capillary, thus significantly enhancing the number of ions that are actually introduced into the mass spectrometer. Triggered by the laser pulse, the PDF unit applies a certain delay (∼µs) before grounding the high voltage supplied to the sample plate, which is then returned to the initial value after several milliseconds. This strategy has shown to improve S/N and signal intensities by a factor of ∼5 over standard AP MALDI conditions (Figure 4a inset). One of the primary advantages of FTMS in protein analysis is the superior mass resolution, which can provide immediate discrimination between isobaric species often encountered during the rapid screening of protein digests typical of proteomics (59) Tan, P. V.; Laiko, V. V.; Doroshenko, V. M. Anal. Chem. 2004, 76, 24622469.

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Figure 5. AP MALDI-FTMS discrimination between the isobaric peptides Leu-enkephalin-Lys (YGGFLK, 684.3721 Da calculated monoisotopic mass for the protonated ion) and Leu-enkephalin-Gln(YGGFLQ, 684.3357 Da calculated monoisotopic mass for the protonated ion), which differ by 0.0364 Da. The analysis was performed in narrow-band mode and provided a resolving power of ∼310 000 M/∆Mfwhm. *, FTMS alias.

investigations. For example, substitution of glutamine for lysine produces peptides that differ by less than 0.04 Da and may require time-consuming derivatization protocols (e.g., preferential acetylation of Lys over Gln) or multiple steps of tandem mass spectrometry (MSn),60 depending on the available mass analyzer. Conversely, AP MALDI-FTMS can immediately discriminate between the isobaric species Leu-enkephalin-Lys (YGGFLK, 684.3721 Da monoisotopic mass) and Leu-enkephalin-Gln (YGGFLQ, 684.3357 Da monoisotopic mass) with no need for lengthy derivatization or separation procedures. The direct high-resolution analysis of a mixture containing both isobaric peptides shows that the two species can be clearly resolved despite a mass difference of only 0.0364 Da (Figure 5). This experiment was performed in narrow-band mode, which provided a resolving power of 310,000 M/∆Mfwhm. CONCLUSIONS The coupling of atmospheric pressure MALDI with Fourier transform mass spectrometry constitutes another important step (60) Bahr, U.; Karas, M.; Kellner, R. Rapid Commun. Mass Spectrom. 1998, 12, 1382-1388. (61) Carroll, J. A.; Penn, S. G.; Fannin, S. T.; Wu, J.; Cancilla, M. T.; Kirk, G. M.; Lebrilla, C. B. Anal. Chem. 1996, 68, 1798-1804. (62) Chaurand, P.; Schwartz, S. A.; Caprioli, R. M. Curr. Opin. Chem. Biol. 2002, 6, 676-681.

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in the evolution of MALDI-FTMS instrumentation. While the earlier introduction of external sources has provided a way to control the kinetic energy of the ions by physically separating the regions where ions are produced and analyzed, AP MALDI has been shown to reduce their internal energy by performing collisional cooling at a much higher pressure than those allowed in standard external sources, which operate under typical vacuum MALDI conditions. Intrinsically cooler ions result in a greatly reduced incidence of metastable decomposition, which translates into larger ion currents available for high-resolution analysis and possible tandem experiments. The ability to rapidly switch the mode of operation at the front end of an atmospheric pressure ionization interface should make the highly complementary MALDI and ESI techniques immediately available to investigators, who are interested in rapid high-resolution analysis of protein digests for protein identification and characterization. Furthermore, the possibility of trapping singly charged ions up to 10 kDa, which was demonstrated in a modified external source MALDI-FTMS,61 suggests that a similar potential should be available for the analysis of intact species with higher molecular masses that exceed the range accessible by ion trap mass spectrometers. In this direction, further work will be necessary to understand and control the declustering process. Finally, the significance of coupling AP MALDI with FTMS extends beyond the preliminary results presented here. Moving the ion generation step out of the vacuum manifold provides the added advantage of eliminating any delay necessary to perform proper evacuation between sample changes, thus increasing the possible analysis throughput. The fact that the spatial distribution of the ions at the moment of desorption does not affect the performance of the analyzer will facilitate the utilization of FTMS in the direct high-resolution analysis of samples present on polyacrylamide gels, membranes, and tissues.62

ACKNOWLEDGMENT D.F. and K.A.K. thank the National Institutes of Health (R01GM643208) for financial support.

Received for review January 27, 2004. Accepted April 21, 2004. AC0498415