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Anal. Chem. 4992, 64, 365-371
Surface- Induced Dissociation of Protonated Peptides: Implications of Initial Kinetic Energy Spread Richard B.
Sylvain LeMeillour: a n d Jean-Claude Tabet*pt
Laboratoire de Chimie Organique Structurale, Universitt?Pierre et Marie Curie, 4 Place Jussieu, 75252 Paris Cedex 05, France, and Department of Chemistry, University of New Orleans, Lakefront, New Orleans, Louisiana 70148
Surfacelnduced dlssoclatlon (SID) has been used to produce daughter ion spectra of small protonated peptides generated by fast atom bombardment (FAB). The relative abundance8 of daughter Ions depends critlcally upon the energy of the lon/surface colllslon. A wlde array of decomposttlon processes may be observed using E M collldon energies In the range 10-20 eV. At approxhnately 1 3 d COlRdon energy,the varlety d decomposltkn processes Is maxhnired for the smal peptkles studied; hence, maxbnun structural Information may be deduced. colllsknally-activated dleeoclatbns (CAD) uslng argon gas and the ldentlcal protonated peptldes could not produce as large an array of daughter Ions In a constant condttlon experiment. An apparent contradlctlon Is thereby posed because SID Is known to produce a narrow dlstributlon of Ion Internal energles relative to CAD. This apparent contradktlon Is resolved by considering the rather large klnetlc energy spread of Ions leaving the FAB source. For the SID process, thls large Initial klnetlc energy dlstrlbutlon Is converted Into a slgniflcantly wlder spread In centerofmass cowskn energy, leading to a broader varlety of decomposition processes (high and low energy) compared to CAD.
INTRODUCTION The promise of mass spectrometry (MS) in peptide sequence analysis has been improved speed and accuracy over conventional wet chemistry methods such as the Edman degradation and other microsequencing methods. Mass spectrometry can overcome certain limitations associated with the Edman method such as the inability to cleave peptides containing a blocked N-terminus or the failure to identify unusual amino acids. The earliest successes applying mass spectrometric methodology to the peptide-sequencingproblem used derivatization to create volatile peptide analogues which were amenable to electron ionization1p2and GC/MS analyses. As new desorption/ionization methods were developed, derivatization was no longer essential and polar, nonvolatile, and thermally-labile compounds such as peptides were shown to be ionized directly by field desorption (FD) mass spectrome t r ~ It . ~was the advent of the particle-induced desorption techniques: particularly fast atom bombardment (FAB),5 however, which opened up the possibilitiesfor routine analysis of small peptides. Although some fragment ions are often present in FAB/MS spectra, structural information can be increased in tandem mass spectrometry experiments6 where selected protonated peptides are subjected to collisional-activation with an inert gas in a field-free region (magnetic mass spectrometer) or an rf-only region (quadrupole mass spectrometer). This FAB/collisionally-activated dissociation (CAD) experiment produces more fragment (daughter) ions
* Authors to whom correspondenceshould be addressed. fUniversit4 Pierre et Marie Curie. t University of New Orleans. 0003-2700/92/0364-0365$03.00/0
that are diagnostic of peptide structure than does conventional MS. In addition, FAB/CAD reduces problems of interfering peaks arising from sample impurities and the liquid matrix. Collision energy, of course, has an important influence upon the character of the CAD spectrum. Under 8-keV collision energy (CAD spectra recorded using magnetic mass spectrometers), a larger variety of fragmentations may occur compared to what is observed for low-energy collisions (CAD spectra obtained near or below 100-eV collision energy with a triple-quadrupole mass spectrometer or BEqQ hybrid instrument). When the energy distribution of collisionally-activated species is considered, the average energy transferred to analyte molecules is approximately the same for the two experiments.’S8 Added peaks in the 8-keV CAD spectrum, however, may be explained by the existence of a low-probability “tail” into the region of high-energy transfer, allowing high-energy decomposition processes to occur. As will be further explored, in the center-of-mass (COM) frame of reference, the maximum energy available for transfer depends upon the mass of the target gas, mt (eq l ) , used to provoke &OM
= ELAB[mt/(mt + mi)]
(1)
decompositions. A heavier target gas atom offers the poesibility that a larger amount of energy may be acquired by the analyte ion. If target gas atoms are substantially lighter than analyte ions, even collisions of higher laboratory energy ( E m ) may produce only a limited array of decomposition processes since only a small fraction of this energy is deposited into the analyte ion. Collision energy in the center-of-mass frame of reference ( E C O M ) represents the maximum quantity of kinetic energy available for conversion into internal energy for an analyte ion of mass mi colliding with a stationary target gas atom. Surface-induced dissociation (SID) is an alternate method for prompting the decomposition of highly stable polyatomic ions, including protonated peptides. This method, whose development was pioneered by Cooks$ relies upon collisions of gas-phase ions with a solid metal (or other) surface to induce ion dissociation. SID offers the potential advantage of an improved collection efficiency for decomposition products especially when compared to low-energy CAD experiments where collision energies may be close to threshold energies and where multiple collision conditions are used, causing ion beam scatter to be particularly high.l0 Additionally, the energy transferred in the center-of-massframe of reference is higher in SID than the analogous transfer during CAD, as surface collisions may be considered to occur between ions and relatively immobile atoms on the metal target (i.e., ECOMis close to Em). The accessibility of high-energy depositions in SID offers the prospect that dissociation of large molecules which are unattainable by CAD may be achieved by SID. Another point of comparison is that SID has been shown to offer a very narrow distribution of energy transferred to colliding polyatomic ions, relative to CAD.” In energy deposition determinations of this type, scattering angle clearly plays an important role. A correlation is known to exist 0 1992 American Chemical Society
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between the angle at which the decomposition ions are detected and the amount of internal energy deposited.12 I t follows that, in CAD experiments, this angular dependence is also a function of the target gas mass. When higher CAD gas pressures are used to achieve multiple collision conditions, the angular dependence of energy transfer is compounded at each collision event, making scattering angle effects from individual collision events difficult to deconvolute. When energy transfer in the SID experiment is compared with that in an angle-resolved CAD experiment, collision energies and scattering angles pertaining to each experiment may be chosen such that the average energy deposition is similar in each case. Under a set of experimental conditions where this was achieved, a narrower distribution of energies was evident for the SID experiment.13 The efficiency of translational-to-vibrationalenergy transfer in SID has been evaluated by Cooks and c o - ~ o r k e r s to ~ ~beJ ~ between 13% and 15% of the laboratory collision energy under the conditions employed, using ELm values ranging from 10 to 90 eV. These results are echoed in recent work by McLafferty, Hunt, and c o - ~ o r k e r swho ’ ~ estimated the efficiency of internal energy deposition in 48-eV (ELAB) SID experiments to be 13% for their set of operating conditions. For the CAD experiment, under single collision conditions in the 5-28-eV @LAB) energy range, Cooks and collaborators8 found the average internal energy uptake for fragmenting (CzH5)4Si’+ ions to vary linearly with E C O M , although the width of the internal energy distribution appears to broaden a t higher collision energies. The slope of the generated curve suggests that, within the employed range of ECoM values, about 7% of each incremental increase in EcoM is converted into internal energy for the colliding ion. At ion kinetic energies below 5 eV, the fraction of &OM transferred into internal energy is higher, although in these CAD regimes, fragmentation is often quite limited. The relatively favorable uptake of vibrational energy inherent to SID allows processes characterized by a much higher activation energy to occur.
EXPERIMENTAL SECTION All SID experiments were performed on a prototype tetraquadrupole MS/MS/MS system.16 A fourth quadrupole was added in front of and at a 90’ angle to the conventional Nermag (Argenteuil,France) R30-10 triple quadrupole. A standard fast atom bombardment (FAB) ion source was situated just in front of the added quadrupole. Selected source ions (in these experiments, FAB-desorbedprotonated peptide molecules) were subjected to collisions at a metal surface located at the intersection of the first and second quadrupole axes. Collision energy was varied by applying an adjustable potential to the ion source and first quadrupole. All reported SID collision energy values represent the differences in potential between the ion source and the metal surface (ELAB).The entire mass spectrometer is differentially-pumped,with a third diffusion pump added to the first quadrupole chamber. The ultimate pressure is approximately Torr, a pressure conducive to rapid coverage of the metal surface with gaseous diffusion pump oil. The first quadrupole was operated manually, while the remaining triple-quadrupole mass spectrometer may be used in all conventional scan modes, permitting the monitoring of consecutive SID/CAD processes. A detailed description of the instrument will be published elsewhere at a later date. FAB/CAD spectra were acquired using the daughter ion (constant parent) MS/MS scan mode of the Nermag R30-10 triple quadrupole in its conventional configuration. Unit resolution was achieved for each quadrupole analyzer under all experimentalconditions. Displayed maw spectra contain all ions having abundances of 1%or more, relative to the base peak. RESULTS AND DISCUSSION The first objective of this study was to find a range of collision energies which gave structurally informative SID spectra of small peptides, while acquiring a qualitative com-
prehension of just how critical the collision energy parameter is toward fragmentation behavior in SID. Of approximately 10 peptides analyzed by FAB/SID, ranging in molecular weight from 200 to 700, without exception, the SID energy yielding the optimum daughter ion spectrum fell in the range 10-20 eV. Outside this energy range, collisions were found to be insufficient (below 10 eV) or too severe (above 20 eV) to give the maximum amount of structural information for peptides of this size. The relative abundances of daughter ions were found to vary significantly even with only a 1-eV change in collision energy. This is demonstrated in Figure 1,which shows the FAB/SID spectra of the peptide GPA (mol wt = 243) taken at 12,13, and 14 eV. The abundances of virtually all fragments are shown to increase relative to MH+ as the collision energy is raised incrementally. The sole exception is m/z 155, which decreases slightly in relative abundance when moving from 13 to 14 eV. This latter result can be rationalized by considering that the 1 eV of additional energy increases the probability that the m/z 155 ions may further decompose via loss of carbon monoxide (mol wt 28, neutral). The concurrent increase in the abundance of the m / z 127 ion suggests this conclusion. It should be noted that it is especially the abundances of the low-mass immonium ions, i.e., m / z 30 (methylimmonium ion) and m/z 70 (from (A2Y21)1cleavage17), which increase with rising collision energy. A second example is that of protonated GGF (Figure 2). Using commonly accepted nomenclature to describe peptide fragmentati~n,’~ at low collision energy (10 eV), Y”-type cleavages ( m / z 166,223) are very prevalent processes, and in general, low-mas ions (e.g., below m/z 100) are present in only minor abundances. At 16 eV, these trends have been virtually reversed; Le., low-mass ions dominate (especially the methylimmonium ion, m/z 301, whereas the relative abundances of Y”-type cleavages have decreased; note also that intact MH+ is no longer detected. These results attest that Y”-type processes (with implicit H-transfer) necessitate lower energy, while m / z 30 formation requires significantly more. It is thus likely that some m/z 30 ions originate from the central glycine or phenylalanine residues from processes involving multiple cleavages which require relatively high activation energies. In contrast, for the intermediate situation (SID collision energy = 13 eV), daughter ions are highly abundant over the entire mass range, an optimum situation for maximizing structural information. Note specifically the ions at m / z 177 and 120, formed by (A3Y2/)2and (A3Y,’l1 processes, respectively, whose relative abundances drop off at both higher and lower collision energies. The 13-eV (center) spectrum in Figure 2, in fact, can be regarded as a type of “weighted average’! of the 10-eV (upper) spectrum and the 16-eV (lower) spectrum. For example, peaks which have high intensities at either the high extreme (16 eV) or the low extreme (10 eV), but are of low intensity (or are absent) at the opposite extreme, have moderate intensities at 13-eVcollision energy. A similar (although less dramatic) trend is evident when the three spectra in Figure 1 are compared. To identify characteristics which might distinguish ionsurface collisions from ion-gas collisions, a comparison was made of FAB/SID spectra with FAB/CAD spectra obtained from identical peptide samples. The chosen model compound is leucine enkephalin which contains five amino acid residues (YGGFL). The enkephalins are known to produce high yields of intact protonated molecules in FABMS.18JQThe decompositions of protonated leucine enkephalin molecules subjected to CAD have been studied in detail by Gaskell et Leucine enkephalin has also been used recently by AberthZ1as a test compound to evaluate the use of a microchannel plate as a collision surface for high-energy SID. In our experiments,
ANALYTICAL CHEMISTRY, VOL. 64, NO. 4, FEBRUARY 15, 1992
244
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280
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1. Suface-induced dissociation (SID) spectra of the protonated peptide GPA. MH+ Ions selected by the fkst quadupole were subjected
166
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1
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to surface collisions using laboratory energies of 12 eV (upper spectrum), 13 eV (center spectrum), and 14 eV (lower spectrum). The second and third quadupoles were operated in the rfonly mode, while the fourth quadrupole was scanned from m / z 10 to 270.
Figure 2. Suface-induceddissociation (SID) spectra of the protonated peptide OGF. MH+ bns selected by the first quadrupole were subjected to surface collisions uslng laboratory energies of 10 eV (upper spectrum), 13 eV (center spectrum), and 16 eV (lower spectrum). The second and third quadrupoles were operated in the rfonly mode, while the fourth quadrupole was scanned from m / r 10 to 300.
various SID collision energies were employed, but 13 eV waa the energy which produced the daughter ion spectrum (Figure 3, SID spectrum) containing the most even distribution of
fragments of MH+ over the entire range of plausible masses (scanning from m / z 10 up past MH+).Included in this spectrum are daughter ions resulting from A-, B-, and Y-type
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ANALYTICAL CHEMISTRY, VOL. 64, NO. 4, FEBRUARY 15, 1992 219
132
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Surface-induced dissociation (SID)spectrum (upper) and collisionalty-activated dissociation (CAD) spectrum (lower)of the protonated peptide leucine enkephalin (YGGFL). The SID energy was 13 eV (ELAB), while the CAD energy was 100 eV (ELAB).The CAD collision Torr (multiple collision gas pressure was approximately 2 X conditions). Figure 3.
cleavages. In addition, the peak at mlz 177 results from (A4Y3’)z internal cleavage, while that at m / z 205 has been assigned to (B4Y3’)*.Note also the presence of the m/z 539 ion, (MH - NH3)+,clearly formed by a lower activation energy process, and again, the methylimmonium ion at-m/z 30, produced by multiple cleavages which presumably require higher activation energies. In a subsequent experiment employing the triple quadrupole in the conventional CAD mode, conditions were sought which would provide a CAD spectrum which was qualitatively similar to this SID spectrum. Using a target gas (Ar)pressure of approximately 2 X Torr (measured just inside the housing of the rf-only quadrupole), and a collision energy of 100 eV (ELAe), the CAD spectrum shown in Figure 3 was obtained. These conditions were chosen because the relative abundances of the immonium ions appearing at m/z 120 and
136 (the two most abundant daughter ions formed by unrelated mechanisms, i.e., (A4Yi)land AI cleavages, respectively) are vitually equivalent to those observed in the SID spectrum (where they are also the two most abundant daughter ions). This likely means that the average vibrational energy uptake for colliding ions is similar for the two types of experiments. This contention is further supported by considering the relative abundances of the mlz 278 and 279 ions formed in the two experiments. These daughter ions are both formed via cleavage of the peptide bond linking the glycine and phenylalanine amino acid residues. Their relative abundances have been shown to vary systematically with collision energy in CAD.=J3 Formation of the m/z 278 ion occurs via B3 cleavage, a higher energy direct cleavage process with charge retention on the fragment containing the amino terminus. On the other hand, formation of the mlz 279 ion occurs by Y2” cleavage, a lower energy procewi requiring hydrogen transfer with charge retention on the fragment containing the carboxy terminus. Since, in each case, rupture of the same peptide bond is occurring, the mlz 278:mlz 279 ratio can be considered as an indicator of relative collision energy in MS/MS experiments; a larger value for this ratio implies a higher average collision energy. In our experiments, the mlz 278:m/z 279 ratio was approximately 2.5 for SID (Figure 3, upper) and approximately 2.3 for CAD (Figure 3, lower). These values are close enough to suggest that the average vibrational energy uptake was not very different for colliding ions in the two experiments. It is evident when the two spectra in Figure 3 are compared, however, that many peaks and, hence, much structural information are absent from the CAD spectrum. These include daughter ion peaks at both the high-mass end (e.g., mlz 539, 425 and the low-mass end (e.g., mlz 30,91, presumably formed by higher energy processes). Certain of these daughter ions may be observed if CAD conditions are altered, but only at the expense of other daughter ions which then disappear. In other words, all the ions shown in the SID spectrum cannot be obtained in one CAD spectrum acquired under constant conditions. It seems appropriate to mention at this point that other researchersz4have been able to show that “collision energy ramping” is a viable method to increase the product ion abundance in CAD experiments. Averaging the daughter ion signals obtained in a series of CAD scans acquired at progressively increasing collision energies yielded a wider array of decomposition processes as compared to the case of fixed-energy CAD experiments. A second example to compare decompositions in SID versus CAD is that of another peptide containing five amino acid residues plus a blocked C-terminus (methyl ester form): HLLVF-OCH,. Conditions for each daughter ion spectrum acquired were identical to the respective conditions used in the previous example. Once again the two most abundant daughter ions (i.e., m / z 110 and 251) are the same for SID and CAD (Figure41, lending further support to the contention that the average uptake of vibrational energy via collision is not very different for the two experiments under the conditions employed which feature quite different collision energies. Here again, the SID spectrum shows more structurally significant daughter ions (mostly A- and B-type cleavages) over a wider mass range (especially high mass, e.g., m / z 463,364) as compared to the CAD spectrum case. SID spectra are thus shown to present a wider variety of daughter ion peaks over a larger mass range than CAD spectra. If, according to quasi-equilibrium theory, the decomposition processes yielding the obtained daughter ion spectra are considered to be a series of competitive and consecutive reactions, then the rate constant for each reaction will vary with the amount of internal energy acquired via collision. A nmow
ANALYTICAL CHEMISTRY, VOL. 64, NO. 4, FEBRUARY 15, 1992 Lower Energy Processes
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Higher Energy Processes
t----.-
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*
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Figure 4. Surface-induced dissociation (SID)spectrum (upper)and cdllsionallyacthrated dtssoclatbn (CAD) spectrum (lower)of the p r e tonated peptide IUVF-OCH,. The SID energy was 13 eV (ELAB), while the CAD energy was 100 eV (ELAB). The CAD collision gas pressure Torr (multiple collision conditions). was approximately 2 X
distribution of acquired internal energies is unlikely to yield a wide variety of decomposition processes except in the rare event that the rate constant curves for many processes cross within this narrow internal energy "window". A broader range of internal energies, of course, increases the likelihood that additional processes may occur. The fact that additional daughter ion peaks appeared in the SID spectra indicates that the range of internal energies acquired by protonated molecules undergoing collisional excitation is largeq for surface collisions (SID,13 eV) than for target gas collisions (CAD, 100 eV). At f i s t glance, this conclusion s e e m to contradict results presented by Cooks et al.'O who showed that a remarkably narrow distribution of internal energies was acquired by EIgenerated (Fe(CO)$+ subjected to SID, as compared to identical samples subjected to CAD. A priori, one possible explanation for this apparent contradiction might be that SID ions having a very wide range of scattering angles are being collected and mass-analyzed. For the instrument employed, the face of the target surface is located at the intersection of the axes of the f i t and second quadrupoles (which are fixed
at 90° to one another). Although a wide range of scattering angles would effectively broaden the spread in energy uptake for colliding ions, the rather large distance between the target and the collection lens leading to the second quadrupole results in a solid half-angle of collection of only 3O. This small angle of collection reduces the likelihood that scattering angle effects play a major role in dictating the distribution of internal energy uptake for mass-analyzed ions. The very sharp tuning required of both the exit lens of the f i t quadrupole, and the entrance optics to the second quadrupole further attest to the likelihood that ions from a rather narrow slice of scattering angles are being analyzed. For these reasons, a large distribution in scattering angles is not believed to be the major factor contributing to the wide distribution in internal energy uptake apparent during SID. Scattering angle effects, however, are likely to play a more important role in widening the distribution of energy uptake during the CAD process. This is especially true since multiple collision conditions were employed, and an rf-only quadrupole collision cell capable of improving the transmission of scattered ions was used in the CAD experiments. The apparent contradiction concerning the widths of internal energy uptake distributions for SID versus CAD may be resolved, however, if one considers the initial kinetic energy distribution of protonated peptide molecules formed by FAB desorption. The initial spread in the translational energies of FAB-desorbed species for our prototype tetraquadrupole, measured in separate e~periments?~ approaches 7 eV (Figure 5). This measured value is slightly higher than a previously reported26kinetic energy range of 5 eV for secondary organic ions desorbed from a liquid matrix or a calculated energy spread value of 3-4 eV (translational and vibrational energy modes) for FAB desorpti0n.2~ In adapting eq 1to these SID and CAD experiments, one can calculate the way in which an initial dutribution of kinetic energies for ions leaving the ion source will affect the distribution of energy available upon collision in the center-ofmass frame of reference. This initial kinetic energy spread translates directly into a distribution of ELAB values when collisions at the respective targets are considered. In the CAD experiment, an initial kinetic energy spread of 7 eV (hELm) is dramatically attenuated when laboratory energy is converted into center-of-mass energy. For a protonated peptide of molecular weight 600 colliding with stationary argon (mt= 40 Da) target gas atoms, an initial spread in laboratory energy of of 7 eV becomes a center-of-massenergy spread @ECOM) about 0.4eV for a single collision event: ~ C O (eV) M = W,.m[mt/(mt + 4 1 = 7[40/(40 + 60011 = 0.4 eV (2) On the other hand, for the SID experiment, where the maximum value of AEcOMapproaches that of A E L ~an, initial kinetic energy spread is virtually fully retained in the cen-
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ANALYTICAL CHEMISTRY, VOL. 64, NO. 4, FEBRUARY 15, 1992
28 1 334
mlz
9
H
111 -L
mlz Flgure 6. Surfacainduced dissociation (SID) spectra of two cyclic peptides. The SID energy was 16 eV (ELAB).
ter-of-massframe of reference. Since virtually no attenuation in AEcoM occurs during SID, the value of AECOM remains close to 7 eV. This relatively enormous energy spread in centerof-mass collision energy for FAB/SID implies that different protonated peptide molecules at opposite extremes of the kinetic energy distribution (Figure 5) will acquire vastly differing amounts of vibrational energy upon collision. This description serve8 to explain the appearance of the larger array of decomposition processes observed in the FAB/SID spectrum. Ions at the high-energy extreme readily decompose via higher energy processes, while those at the lower energy end are limited to low-energy pathways. Even though multiple target gas collisions will widen the spread in internal energy uptake for colliding ions in the FAB/CAD experiment, the internal energy distribution is evidently still not as wide as in FAB/SID. The preceding arguments concerning the consequences of an initial kinetic energy distribution for ions subjected to the SID process should be valid regardless of the means employed to produce the ions. Other ionization methods such as electron ionization and chemical ionization clearly offer a lower spread in kinetic energies for ions leaving the source compared to that of FAB ionization. The resultant distribution in internal energies acquired via surface collision for EI- or CI-generated ions is thus expected to be correspondugly narrower than that for FAB. As the size of the analyte ion increases, the [mt/(m,+ mi)] term in eq 1becomes smaller for the CAD experiment. This fact has been used, along with other arguments concerning the increased number of modes available for internal energy deposition in larger molecules, to explain the difficulty which is experienced in obtaining CAD data on peptides having molecular weights above 2500. Relating this information to our experiments, as the molecular weight of the analyte ion increases while other variables are held constant, ECOM values for CAD and SID should diverge, since ECoM drops off for the former experiments, while it remains more or less constant for the latter. The same statement can be made for AECOM values. Higher E C o M values lend hope to the possibility of obtaining higher daughter ion yields for SID of larger peptides
than have been achievable via CAD. Wider AEcoM values offer the possibility that a larger array of processes could be observed in a constant condition SID experiment. In the case of very large molecules, however, it is possible that even a 7-eV value for A&OM is still too small to permit many different types of fragmentation processes. Thus, eventually, collision energy ramping for SID may be required to observe a large array of structurally informative daughter ions from very large parent molecules. While instrumental constraints prevent the direct testing of these hypotheses due to the mass range limitations of the quadrupole analyzers used in this study, a final pair of examples can further promote the argument that peptides of higher stability can be quite readily analyzed by SID. The examples are those of two similar cyclic peptides appearing in Figure 6. Cyclic peptides are, of course, difficult to analyze by conventional sequencing methods which require an unblocked N- or C-terminus. The cyclic structure necessitates the cleavage of at least two bonds to produce relevant sequencing information in tandem mass spectrometry experiments. For linear peptides, of course, sequence information can be obtained from ions formed via rupture of a single bond. Figure 6 clearly demonstrates the ability of 16-eV collisions to provide ample energy to provoke a wide variety of daughter ions bearing sequence information for these “higher stability” peptides.
CONCLUSION SID can offer certain advantages over CAD as a tandem mass spectrometricanalysis method. Among its distinguishing features are an increased propensity for collision, a single collision interaction, and the potential for improved sensitivity under conditions where scatter is minimized. The kinetic energy of ions impacting the metal surface is extremely critical in determining which types of daughter ions will be formed during the SID process. Small changes in ELmfor colliding ions will be converted rather directly into E C o M differences, leading to substantially altered SID spectra. Comparable changes in ELABduring CAD experiments have much more subtle effects on obtained spectra. An initially broad range of ion kinetic energies will result in widely variable uptakes of internal energy in SID experiments. When an ionization technique such as FAB is used, which imparts a large kinetic energy spread onto ions leaving the source, 13-eV SID spectra of small protonated peptides contain a mixture of fragment ions originating from both low- and high-energy processes. These same daughter ions cannot all be produced under a single CAD condition. Structural information can thus be maximized for small peptides in a constant condition SID experiment. Since very low SID collision energies (e.g.,
