Fragmentation of Peptides in MALDI In-Source Decay Mediated by Hydrogen Radicals

Transcription

1 Fragmentation of Peptides in MALDI In-Source Decay Mediated by Hydrogen Radicals Thomas Ko1 cher,*, Åke Engstro1 m, and Roman A. Zubarev Laboratory for Biological and Medical Mass Spectrometry, Uppsala University, Uppsala, Sweden In-source decay (ISD) in matrix-assisted laser desorption/ ionization (MALDI) shares some similarities with the novel fragmentation technique electron capture dissociation (ECD). In both reactions, the otherwise strong N-C r bond is cleaved, forming fragment ions of the c and z types, while labile posttranslational modifications are preserved. Therefore, it is tempting to assume that ISD and ECD have some mechanistic aspects in common. Because electrons are present in the MALDI plume, we investigated the previously suggested possibility that ISD is a variation of ECD. However, experiments with peptides with only one site for efficient protonation revealed that ISD is not caused by electron capture. Instead, ICD seems to be induced by hydrogen atoms generated by a photochemical reaction of the matrix. We provide evidence for this reaction by hydrogen/deuterium exchange experiments with peptides containing a minimal number of exchangeable hydrogen atoms. The hydrogen atom model in ECD is indirectly supported by the proposed fragmentation mechanism for ISD, because our data suggest that hydrogen radicals can induce fragmentation by cleavage of the N-C r bond, independent from their origin. The invention of soft ionization techniques 1,2 capable of ionizing peptides and transferring them to the gas phase at a high efficiency and without substantial fragmentation was the crucial prerequisite for the recent dramatic advances in protein analysis. 3-5 In the most widely used strategies, the proteins are first cleaved by a proteolytic enzyme, followed by mass spectrometry-based * Phone: Fax: Thomas.Kocher@ bmms.uu.se. Laboratory for Biological and Medical Mass Spectrometry, Uppsala University, Husargatan 3, Box 583, Uppsala, SE-75123, Sweden. Department for Medical Biochemistry and Microbiology, Uppsala University, Husargatan 3, Box 582, Uppsala, SE-75123, Sweden. (1) Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse, C. M. Science 1989, 246, (2) Karas, M.; Hillenkamp, F. Anal. Chem. 1988, 60, (3) Gavin, A. C.; Bosche, M.; Krause, R.; Grandi, P.; Marzioch, M.; Bauer, A.; Schultz, J.; Rick, J. M.; Michon, A. M.; Cruciat, C. M.; Remor, M.; Hofert, C.; Schelder, M.; Brajenovic, M.; Ruffner, H.; Merino, A.; Klein, K.; Hudak, M.; Dickson, D.; Rudi, T.; Gnau, V.; Bauch, A.; Bastuck, S.; Huhse, B.; Leutwein, C.; Heurtier, M. A.; Copley, R. R.; Edelmann, A.; Querfurth, E.; Rybin, V.; Drewes, G.; Raida, M.; Bouwmeester, T.; Bork, P.; Seraphin, B.; Kuster, B.; Neubauer, G.; Superti-Furga, G. Nature 2002, 415, (4) Mootha, V. K.; Bunkenborg, J.; Olsen, J. V.; Hjerrild, M.; Wisniewski, J. R.; Stahl, E.; Bolouri, M. S.; Ray, H. N.; Sihag, S.; Kamal, M.; Patterson, N.; Lander, E. S.; Mann, M. Cell 2003, 115, (5) Schirmer, E. C.; Florens, L.; Guan, T.; Yates, J. R., 3rd; Gerace, L. Science 2003, 301, analysis. In this so-called bottom-up approach, either the peptide mixtures are then analyzed with tandem mass spectrometry or the protein is identified on the basis of peptide mass fingerprinting with matrix-assisted laser desorption/ionization (MALDI) timeof-flight (TOF) mass spectrometry. 2 In both approaches, it is essential for the sensitivity of the analysis and for the identification process that ionization occur without intrinsic fragmentation. Although MALDI was initially believed to be a nondestructive ionization technique, 2,6 it became apparent that a significant degree of fragmentation is associated with the ionization event. 7-9 This decay is observed either as an in-source decay 7,8 (ISD) or during mass separation as a post-source decay process 9 (PSD). Both ISD and PSD have become widely used for obtaining the primary sequence of peptides. In contrast to PSD, ISD can be used for sequencing of much larger peptides and even proteins. 7,10 On the other hand, ISD of small peptides proved to be difficult, because the low m/z region is obscured by matrix clusters, and the yield of fragmentation in ISD is generally lower for small peptides. Logically, the most common application of ISD is found in topdown approaches to protein characterization. 10,11 These emerging approaches to protein identification are based on the analysis of intact proteins 12,13 instead of their proteolytic peptide mixtures. A vast majority of the reported methods utilize highly charged ions generated by electrospray ionization. Although top-down mass spectrometry is still limited by protein size and sample complexity, new and improved mass spectrometry instrumentation and the invention of electron capture dissociation 14,15 (ECD) make this approach a tempting alternative to traditional peptide-based methods. ECD with the associated increased number of cleavage sites proved to be an important technique to achieve the required complete sequence coverage. 16 However, as mentioned above, a (6) Karas, M.; Bachmann, D.; Bahr, U.; Hillenkamp, F. Int. J. Mass Spectrom. Ion Processes 1987, 78, (7) Brown, R. S.; Lennon, J. J. Anal. Chem. 1995, 67, (8) Brown, R. S.; Feng, J.; Reiber, D. C. Int. J. Mass Spectrom. Ion Processes 1997, 169/170, (9) Spengler, B.; Kirsch, D.; Kaufmann, R.; Jaeger, E. Rapid Commun. Mass Spectrom. 1992, 6, (10) Reiber, D. C.; Grover, T. A.; Brown, R. S. Anal. Chem. 1998, 70, (11) Suckau, D.; Resemann, A. Anal. Chem. 2003, 75, (12) Reid, G. E.; McLuckey, S. A. J. Mass Spectrom. 2002, 37, (13) Ge, Y.; Lawhorn, B. G.; El Naggar, M.; Strauss, E.; Park, J. H.; Begley, T. P.; McLafferty, F. W. J. Am. Chem. Soc. 2002, 124, (14) Zubarev, R. A.; Kelleher, N. L.; McLafferty, F. W. J. Am. Chem. Soc. 1998, 120, (15) Zubarev, R. A.; Horn, D. M.; Fridriksson, E. K.; Kelleher, N. L.; Kruger, N. A.; Lewis, M. A.; Carpenter, B. K.; McLafferty, F. W. Anal. Chem. 2000, 72, (16) Horn, D. M.; Ge, Y.; McLafferty, F. W. Anal. Chem. 2000, 72, /ac CCC: $30.25 xxxx American Chemical Society A Published on Web 00/00/0000 PAGE EST: 5.8

2 much simpler and faster MALDI-TOF analysis at elevated laser fluence can be used alternatively for primary structure elucidation of large sequence stretches of intact proteins by exploiting insource fragmentation. 10,17 The applicability of the method is limited to the analysis of homogeneous samples at high quantities, because ISD does not allow selection of a precursor ion and generates a low yield of product ions. The mechanism and the time frame of PSD and ISD are extremely different. PSD is thought to be mostly due to bimolecular collision processes. The observed fragment ions, primarily of the a- and b-type ion series, can be explained mainly by vibrational activation processes. 18 The optimal MALDI matrix for PSD is R-cyano-4-hydroxycinnamic acid (CHCA), but the same matrix leads to poor fragmentation in ISD. 8 The fragmentation in PSD occurs in the field free region of a reflectron TOF mass spectrometer. ISD products can be easily distinguished from PSD ions by their much shorter fragmentation time frame. 8,19 This very fast formation of ISD ions results in the useful feature that the fragment ions have the same kinetic energy as the peptide ions at the starting point of the ion separation in the TOF analyzer. As a consequence, the fragment ions are focused and resolved, and their time-dependent separation can be calibrated by the same calibration curve as for the intact peptide ions. Coincidently, there seems to be an additional connection between ISD and ECD apart from their use in the attempts to characterize intact proteins in top-down approaches. In both techniques the otherwise strong N-C R bond is cleaved, resulting in c- and z-type fragments. 7,14 These main fragmentation products are accompanied by minor peaks corresponding to y- and a-ions. In ISD, the ratios between the abundance of these ion series strongly depend on the MALDI matrix. 8 Similar to the MALDI process itself, the mechanism of ISD is not completely understood Recently, electron-ion reactions of multiply charged MALDI ions were proposed 21,23 as a possible route for ISD fragment formation. This hypothesis was substantiated by the reported presence of electrons in the MALDI plume; very fast fragmentation kinetics; 8,19 preferential cleavage of S-S bonds; 27 similar fragmentation patterns; and the important common characteristics that labile modifications, such as phosphorylation 28 or sulfatation, 29 are preserved in both fragmentation techniques. However, the topic remained controversial, because several lines of experimental evidence, such as the generation of ISD ions in negative mode 8 and the high dependence of the yield, and the nature of ISD fragments on the used matrix 8 are difficult to explain with an ECD-based mechanism. The aim of this study was to investigate the mechanism of ISD from the viewpoint of a comparison between ECD and ISD, (17) Katta, V.; Chow, D. T.; Rohde, M. F. Anal. Chem. 1998, 70, (18) Chaurand P.; Luetzenkirchen F.; Spengler B. J. Am. Soc. Mass Spectrom. 1999, 10, (19) Takayama, M. J. Am. Soc. Mass Spectrom. 2001, 12, (20) Karas, M.; Kruger, R. Chem. Rev. 2003, 103, (21) Karas, M.; Gluckmann, M.; Schafer, J. J. Mass Spectrom. 2000, 35, (22) Knochenmuss, R.; Zenobi, R. Chem. Rev. 2003, 103, (23) Zubarev, R. A.; Haselmann, K. F.; Budnik, B.; Kjeldsen, F.; Jensen F. Eur. J. Mass Spectrom. 2002, 8, (24) Gorshkov, M. V.; Frankevich, V. E.; Zenobi, R. Eur. J. Mass Spectrom. 2002, 8, (25) Frankevich, V.; Knochenmuss, R.; Zenobi, R. Int. J. Mass Specrom. 2002, 220, (26) Knochenmuss, R. Anal. Chem. 2004, 76, (27) Patterson, S, D.; Katta, V. Anal. Chem. 1994, 66, (28) Lennon, J. J.; Walsh, K. A. Protein Sci. 1999, 8, (29) Takayama, M.; Tsugita, A. Electrophoresis 2000, 21, starting with the hypothesis that ISD is a variation of ECD. However, here we will show that ISD cannot be caused by an electron-capture event by the analyte cations. Additionally, we will present novel evidence that ISD is a radical reaction resulting in the same reaction products as ECD. In addition, we will show by using hydrogen/deuterium exchange experiments that cleavage in ISD is mediated by hydrogen radicals photochemically generated from the MALDI matrix, as has been suggested recently. 30 Understanding the origin of ISD might allow for improvements of the current methodologies to increase the product ion yield of ISD. EXPERIMENTAL SECTION Materials. Synthetic peptides were synthesized using a solidphase synthesizer (Intavis, Germany) according to the manufacturer s instructions. All solvents used were of HPLC grade. Acetic anhydride, methanol, acetyl chloride, 2,5-dihydroxybenzoic acid (DHB), and R-cyano-4-hydroxycinnamic acid (CHCA) were purchased from Merck (Darmstadt, Germany). HPLC grade water was bought from Aldrich Chemical Co (Milwaukee, WI). NH 4 HCO 3 was purchased from Sigma Chemical Co (St. Luis, MO). C18-ZipTips were obtained from Millipore (Billerica, MA). Chemical Modifications. For the acetylation of the N terminus and lysine residues, peptides were incubated with acetyl anhydride at a 1: molar excess at 29 C.A1-µL portion of 5% acetyl anhydride in H 2 O was added to the peptide dissolved in 18 µl of0.1mnh 4 HCO 3. After 20 min of reaction time, the procedure was repeated by adding an additional aliquot of acetyl anhydride. Diluted NH 3 was added to keep the reaction mixture above ph 8. Methyl-esterification of peptides was performed with a 1.5:10 mixture of acetyl chloride in water-free methanol. 31 A 15-µL portion of acetyl chloride was then added to 100 µl of methanol at -20 C. After 15 min of incubation, 15 µl ofthe mixture was added to the dried peptide sample. The reaction mixture was allowed to react for 45 min at room temperature and was subsequently dried down in a vacuum centrifuge. Prior to mass spectrometry, the peptides were desalted using ZipTips (Millipore) according to the manufacturer s recommendations. Deuterium labeling was achieved by on-target exchange of the prepared MALDI sample with D 2 O. Sample Preparation. Samples were prepared using the dried droplet method with 2,5-DHB as the matrix as described 2 by mixing 0.5 µl of the analyte solution with 0.5 µl of a 20 mg/ml solution of 2,5-DHB in 50% ACN/50% H 2 O. In all experiments, a 10 µm solution of the peptide was used, resulting in the deposition of 5 pmol of peptide on the target. The mixture was spotted on the metal target and was allowed to dry. In experiments studying the effect of photoelectrons emitted from the metal target, the surface was covered with a layer of adhesive tape. MALDI Mass Spectrometry. All MALDI spectra were acquired on a Ultraflex TOF/TOF (Bruker, Germany) equipped with a gridless delayed extraction ion source, a 337-nm nitrogen laser, and a gridless ion reflector. ISD spectra from the peptides were acquired in the reflector mode, using a 25-kV acceleration voltage and a 150-ns ion extraction delay. The low-mass ion deflector cuttoff was set to 350 Da. For ISD experiments, the laser fluence (30) Takayama, M. J. Am. Soc. Mass Spectrom. 2001, 12, (31) Hunt, D. F.; Yates, J. R., 3rd; Shabanowitz, J.; Winston, S.; Hauer, C. R. Proc. Natl. Acad. Sci. U.S.A. 1986, 83, B

3 Scheme 1 Scheme 2 Figure 1. The ISD spectrum of the peptide LRRLEMYCAPLKPAK is shown. Panel A: The doubly and singly charged peptide ion and some c + - and z + -ions are indicated. The peaks resulting from the cleavage at the cysteine residue, corresponding to the [z 8 - SH ] + ion and the [a 8 - SH ] + are labeled. Panel B: A subset of the ISD spectrum is shown. The peaks corresponding to the products of radical reactions of the initially formed radical of the z-ion series are indicated. The ions undergo either loss of a hydrogen atom or gain of a hydrogen atom or react with a matrix radical to an ion 153 Da higher in mass. was raised to 10% above threshold. The obtained spectra were interpreted manually using flexanalysis 2.0 (Bruker, Germany). MALDI spectra were calibrated externally with a mixture of standard peptides. Nomenclature. We used an extension to the traditional nomenclature 32 described previously. 33 The presence of an unpaired electron is always denoted by a radical sign. A hydrogen transfer to the radical is indicated by the prime sign, such as in c. The loss of a hydrogen atom from the homolytic cleavage product ion c would be described by c +. RESULTS AND DISCUSSION ISD of Peptides: a Radical Reaction Leading to ECD-Type Fragment Ions. The ISD spectrum of the peptide LRRLEMY- CAPLKPAK was acquired by performing MALDI TOF mass spectrometry on a dried droplet preparation with 2,5-DHB at elevated laser fluence (Figure 1). The fluence used depended on the spatial concentration distribution of the analyte and on the crystal shape, but was set 10% higher than the threshold for ion formation. The combined yield of fragmentation products in this experiment was in the range of 10%, normalized on the peak intensity of the protonated molecular species. Although 2,5-DHB has been reported to be the matrix most suited for ISD, 8,30 this relatively low efficiency of ISD was generally observed in all of our experiments, thus limiting the applicability of ISD to sample quantities in the low picomole range. The obtained ISD spectrum (32) Roepstorff, P.; Fohlman, J. Biomed. Mass Spectrom. 1984, 11, 601. (33) Kjeldsen, F.; Haselmann, K. F.; Budnik, B. A.; Jensen, F.; Zubarev, R. A. Chem. Phys. Lett. 2002, 356, displayed a variety of different fragment ion series and peaks corresponding to secondary fragmentations (Figure 1, panel A). The observed c-, z-, y-, and a-ions were similar to the type of fragment ions usually observed with ECD. A direct comparison of the relative intensities of the fragment ions in both methods revealed differences which might be explained by the different instrumentation and by other reactions during the MALDI process. Similarly to ECD, ISD fragmentation occurs on a very fast time scale. Consequently, changing the delay time from 300 to 30 ns did not reduce the ion yield nor the fragment ion resolution and the overall pattern of fragmentation significantly (data not shown). In ECD, it is hypothesized that the mechanism of fragmentation is mediated by a reactive hydrogen atom generated by the electron capture event, generating pairs of c + - and z -ions (Scheme 1) or as a minor fragmentation channel y + - and a -ions. 15 The radical site is present in one of the produced fragments, usually in the z- or the a-ion series. 14 In contrast to ECD, ISD is not a tandem mass spectrometry technique and does not allow for precursor ion selection; therefore, contaminations in the sample might be misinterpreted as fragment ions. In this context, it should be noted that the masses of y + -ions are identical with truncated versions of the peptide that might be present even after HPLC separation. Additionally, y + -ions can be generated by other activation processes, such as vibrational activation. Logically, conclusions should not be based solely on interpretation of these putative y + -ions. For this reason, we focused in our investigation on the unique c- and z-ion series. In contrast to ECD, all z-ions observed in ISD are even electron species, a serious discrepancy between the experimental data and our initial hypothesis rationalizing ISD as an ion-electron reaction. However, the detailed analysis of the ISD fragment ions showed that these species can be explained as radical ions that either lost or gained a hydrogen atom or reacted with a matrix radical, generating an adduct 153 Da higher in mass than the proposed radicals initially formed in the cleavage reaction (Figure 1, panel B and Scheme 2). The reaction with a hydrogen radical leading toaz + ion seemed to be the dominant reaction route because this peak had the highest intensity in the spectra of all peptides we analyzed, but the relative intensities depended on the primary structure of the peptide, and we observed a low reproducibility of the exact pattern, even with the same peptide. As predicted by C

4 Scheme 3 the model for fragmentation induced by a hydrogen mediated radical cleavage, 15 adducts generated by the recombination of ISD radicals with matrix radicals were not present in the c-ion series or in the y-ion series. These findings confirmed the nature of ISD as a radical reaction, giving rise to the same type of initial fragment ions as in ECD. The nature of the fragment ions observed with ISD strongly depended on the distribution of basic residues within the peptide and the stability of the generated fragments. In the analysis of the peptide LRRLEMYCAPLKPAK, we observed all possible ion series, most probably because basic residues are present at both termini of this peptide. Similarly to ECD, the N-C R bond was cleaved preferentially, resulting in higher combined intensities of the c- and z-ions when compared to the y- and a-ions. The c + -ion series was found to be dominant, most likely because the stability of the c + -ions is higher than that of the radical z-ions, which can undergo a variety of radical reactions. Interestingly, the z 8 + product ion arising from a cleavage adjacent to the cysteine residue was of low intensity in this spectrum. At the same time, we detected a very intense signal corresponding to z 8, which lost the SH group (Figure 1, panel A). Formation of this ion has already been reported for ECD of alkylated cysteine containing residues. 34 Additionally, the a 8 -ion terminating at the cysteine residue, which lost the SH, group was detected with a very high intensity. Both [z 8 - SH ] + and [a 8 - SH ] + ions were present at a much higher intensity than other ions of the respective ion series. Consequently, the c 8 + -ion, resulting from N-C R cleavage at the same position, was found to be higher in intensity when compared to the adjacent ions of the c-ion series. Alkylation of the cysteine did not change the relative intensities of these fragments. The identity of these product ions was confirmed by mutating amino acids N- and C-terminal to the cysteine residue within the peptide. These mutations in all cases lead to the expected mass shifts (data not shown) without significant changes in the intensity of the corresponding fragment ions. Furthermore, the products resulting from recombination of a radical ion with matrix radicals were absent in these spectra. Both the [z 8 - SH ] + or the [a 8 - SH ] + ion can be explained by R-cleavage in the z 8 or the a 8 radical ion initially generated by fragmentation of the N-C R bond (Scheme 3a). 34 However, given the high hydrogen (34) Mormann, M.; Macek, B.; de Peredo, A. G.; Hofsteenge, J.; Peter-Katalinic, J. Int. J. Mass Spectrom. 2004, 234, affinity of sulfur, we propose a different reaction pathway (Scheme 3b) starting with the attack of the hydrogen radical at the sulfur functionality, similar to the route proposed for the cleavage of monosulfide bridges in ECD of lantibiotics. 35 The very high intensity of the [a 8 - SH ] + ion can be better rationalized by this reaction, given the very weak abundance of all the other ions in the a-ion series. The combined results of the peptide LRRLEMY- CAPLKPAK strongly support the hypothesis that ISD is a radical reaction, following a fragmentation mechanism similar to the hydrogen atom model in ECD. Role of Photoelectrons. As mentioned above, it has been proposed that ISD is driven by electron capture of peptides during the MALDI ionization event. 21,23 One possible source of these electrons could be photoemission from the matrix-metal interface of the MALDI target, 36 which has been reported to be responsible for an electron capture driven ISD process. 37 It became evident from our own data and the reported results of other groups 19 that 2,5-DHB gives a much better yield of ISD ions than the CHCA matrix does. Interestingly, CHCA leads to a higher yield of doubly charged ions, which should, in principle, promote ECD-type fragmentation. 38 Additionally, dried droplet preparations with 2,5- DHB generally result in thicker crystal structures, as compared to CHCA, which should again favor CHCA as a better matrix for ISD, because thicker crystals generate little photoelectrons from the matrix-metal interface. 36 This seems contradictory to the hypothesis that photoelectrons captured by doubly charged ions should be the cause of ISD, because in this case, CHCA should be a better matrix for ISD when compared with 2,5-DHB. To clarify this discrepancy, we investigated the role of electrons emitted from the metal surface by isolating the surface of the metal target with an adhesive tape. The resulting preparation gave similar ISD spectra, as compared with a MALDI preparation on a clear metal surface. This suggests that at least in the case of 2,5-DHB, photoelectrons do not play a role in the process. Next, we analyzed the ISD fragmentation of substance P using CHCA as a matrix. In the spectrum recorded under ISD conditions, we observed for the metal target preparation a c + -ion series and the metastabile PSD type of ions characteristic for CHCA. 9 However, in contrast to previous observations, 37 we still observed ISD ions with the adhesive tape covering the metal surface, suggesting that photoelectrons from the interface of a metallic surface are at least not the only cause of ISD fragmentation in the case of CHCA. It was impossible to compare the yield of ISD fragmentation in the different preparations because of the parameters changing not related to the emission of photoelectrons, such as the smaller spot size in the preparation using the adhesive tape. ISD of Peptides with a Single Protonation Site. These results do not rule out the possibility that ISD is mediated by electron capture, because photoelectrons emitted from the metal surface are not the only potential source of electrons present in the MALDI process. 21 If ISD fragmentation is generated by an electron capture event, the charge state of the capturing species (35) Kleinnijenhuis, A. J.; Duursma, M. C.; Breukink, E.; Heeren, R. M.; Heck, A. J. Anal. Chem. 2003, 75, (36) Frankevich, V. E.; Zhang, J.; Friess, S. D.; Dashtiev, M.; Zenobi, R. Anal. Chem. 2003, 75, (37) Frankevich, V.; Zhang, J.; Dashtiev, M.; Zenobi, R. Rapid Commun. Mass Spectrom. 2003, 17, (38) Beavis, R. C.; Chaudhary, T.; Chait, B. T. Org. Mass Spectrom 1992, 27, D

5 Scheme 4 Figure 2. The ISD spectra of substance P (panel A), acetylated substance P (panel B), and their equimolar mixture (panel C) are shown. Panel A: the spectrum of substance P is given as a control. The doubly charged peptide ion and the c + -ion series are indicated. Panel B: The spectrum of acetylated substance P demonstrates that a singly charged ion can undergo ISD. The c + -ion series is labeled, and the position of the missing doubly charged peptide ion is indicated with an arrow. Panel C: ISD of the equimolar mixture demonstrates that ISD of the acetylated peptide is of an efficiency similar to that of the unmodified peptide. The mass shift of two times 42 Da is indicated with an arrow. must be higher than one, because otherwise, the generated fragments would be neutral species and could not be detected with mass spectrometry. This is independent of the actual mechanism of fragmentation and does not depend on the validity of current models for ECD. However, it seems to be impossible to determine the precise nature of the fragmenting species in ISD, such as its charge state, because fragmentation and ionization are closely linked in time for ISD, and selection of a defined precursor ion is not possible. Consequently, we investigated the nature of ISD by analyzing a peptide that is limited to a maximum charge state of one. We measured the ISD spectrum of the acetylated peptide acetyl-rp[acetyl-k]pqqffglm (acetylated substance P with a free carboxy terminus). In this peptide, the guanidino group of the arginine residue is the single site for efficient protonation. As a control, we acquired the ISD spectrum of substance P. The spectrum of the unmodified peptide (Figure 2, panel A) was dominated by the c + -ion series, showing an even distribution of the abundances of the fragment ions, except for the intensity of the peak of c 8 + arising from cleavage N-terminal to the glycine residue. The relatively low abundance of the c + fragment ions N-terminal to glycine was a general observation for the ISD spectra of proteins and peptides. The peak corresponding to the doubly charged ion was present at an intensity similar to that of the ISD ions. Next, we analyzed the reaction product of substance P with acetyl anhydride under MALDI ISD conditions. It should be noted that the doubly charged peak present in the spectrum of substance P was not detected with the acetylated peptide (Figure 2, panel B). Given the single protonation site of this peptide, the ISD ions of this peptide should be absent in a similar fashion if ECD is the reason for ISD. Intriguingly, the overall character of the spectrum was similar to that of the unmodified peptide, but with the N-terminal ion series shifted by 84 Da, corresponding to the two acetylation sites in the peptide (Figure 2, panel B). These results strongly suggested that ISD is not mediated by a direct electron capture of the peptide. Theoretically, a singly charged peptide could capture an electron, fragment, and subsequently be recharged by a charge-transfer process in the MALDI plume. This scenario is very unlikely because doubly charged peptides have a much higher probability to capture an electron than do singly charged peptides because the capture cross section is proportional to the ionic charge squared (z 2 ). 15 Therefore, even in a chargetransfer scenario, the acetylated peptide should generate a greatly reduced number of ISD ions in the case of an electron-capturemediated fragmentation. For additional clarity, we recorded an ISD spectrum of a mixture of acetylated substance P mixed with an approximately equal amount of unmodified substance P in order to obtain precise relative signal intensities. In the obtained spectrum, the intensities of the c-ion series of both peptides were almost equal (Figure 2, panel C), ruling out the possibility of a mechanism including a charge transfer onto a neutral fragment. These results proved that ISD is not mediated by electron capture of doubly charged peptide ions. H/D Exchange Experiments. To investigate the nature of the source of the hydrogen atom involved in the ISD fragmenta- E

6 Figure 3. H/D exchange experiment with the methyl esterified peptide [Sar] 13-Pro. Panel A: The MH + ion of the methyl esterified peptide is shown. Panel B: Labeling with deuterium resulted in a mass shift of 2 Da, corresponding to a H/D exchange at the N terminus and the charging proton. Panel C: The c 11 + ion of the ISD spectrum of the methyl esterified peptide is shown. Panel D: The c 11 + ion generated by the deuterium labeled sample is shifted by 3 Da. tion, we analyzed the peptide [sarcosine] 13 -proline (Scheme 4). This peptide was chosen to simplify the interpretation of the H/D exchange experiment because this peptide does not contain exchangeable hydrogen atoms at the amide bonds of the peptide backbone. First, we confirmed that this peptide has only one site for efficient protonation by analyzing its acetylated form. The N-terminal acetylated peptide should not be detectable in its protonated form because the single protonation site was removed by acetylation of the free amino group. Hence, the corresponding MALDI spectrum was dominated by the sodium and the potassium adducts of the acetylated species (data not shown). Any signal corresponding to the protonated acetylated peptide was absent in the spectrum, proving that there is only one protonation site in the unmodified peptide. Although only singly charged, the unmodified peptide showed a complete c + -ion series with ISD. This result was in line with the previous results obtained with the acetylated form of substance P. In the following H/D experiment, we analyzed the methyl esterified derivative of this peptide containing, in addition to the charging proton, only one exchangeable hydrogen atom at the N terminus of the peptide. After recording the ISD spectrum of the peptide (Figure 3, panels A and C), we incubated the MALDI preparation with D 2 O, repeated the labeling procedure several times, and recorded another ISD spectrum (Figure 3, panels B and D). After labeling with deuterium, the mass of the peptide (m/z ) was shifted by 2 Da(m/z ), as expected from the sequence of the peptide. In contrast to the peak of the singly charged peptide ion, the c + -ions, although containing only one exchangeable hydrogen and one charging proton, were all shifted by 3 Da (Figure 3, panels C and D). This suggests that the additionally transferred deuterium is from the deuterated matrix (Scheme 4). The spectrum of 2,5-DHB was recorded with very low laser fluence and showed an expected incorporation of three deuterium atoms. Repeating the labeling experiment using the unmodified peptide (data not shown), we observed a mass shift of 3 Da for the peptide ion. This was expected because the peptide contains, in addition to the charging proton, two exchangeable hydrogen atoms located at the N and C termini, respectively. The c + -ions, although containing only one exchangeable hydrogen in addition to the charging agent, were all shifted by 3 Da. The combined data suggested that the transferred hydrogen is from the deuterated matrix, probably by a photochemically induced mechanism. CONCLUSION We clearly showed that ISD is not mediated by direct electron capture of peptides. Our data supports a fragmentation mechanism similar to the hydrogen atom model previously suggested to explain fragmentation in ECD. We suggest that the reactive hydrogen atom in ISD is generated by the matrix, possibly as a result of a photochemical reaction. Because this electron-free fragmentation in ISD can be rationalized by a hydrogen atom model similar to ECD, the obtained data give strong, although indirect, support for the hydrogen model for ECD. ACKNOWLEDGMENT We thank other members of our laboratories for help and fruitful discussions and Christopher Adams for critical reading of the manuscript. The work in R.Z. s and A.E. s laboratories was supported by the Wallenberg Consortium North. T.K was a recipient of a postdoctoral fellowship of the European Molecular Biology Organization (EMBO). Received for review July 26, Accepted October 8, AC F PAGE EST: 5.8