Anal. Chem. 1996, 68, 522-526
Correlation of Kinetic Energy Losses in High-Energy Collision-Induced Dissociation with Observed Peptide Product Ions Richard W. Vachet, Andrew D. Winders, and Gary L. Glish*
Department of Chemistry, CB 3290, University of North Carolina, Chapel Hill, North Carolina 27599-3290
In collision-induced dissociation, some of an incident parent ion’s kinetic energy is converted into internal energy upon collision with a neutral target. The kinetic energy lost is related to the amount of internal energy deposited into any individual ion. To see dissociations of different critical energies on the same time scale, different amounts of internal energy need to be deposited. This should be reflected in the kinetic energy lost by the parent ion in the formation of different product ions. Variable amounts of energy loss in the formation of different peptide product ions are reported here. It is seen that different product ion types (b, y, a) show ordered patterns of energy losses. A greater energy loss is observed for the formation of b-type product ions than for y-type, and even greater energy losses are observed for the formation of a-type product ions. A very good correlation between ion type energy loss and ion mass is observed. Thus, measuring the energy losses in the formation of product ions may provide a means for classifying the product ion type. Tandem mass spectrometry (MS/MS) has become a powerful method for structural analysis of biomolecules. In particular, much interest has been paid to elucidating the primary structure of peptides.1,2 The dissociation of peptide ions has typically involved collision-induced dissociation (CID) of the protonated species [M + H]+.2,3 CID is usually described as occurring in two steps: ions are activated by the conversion of some of their kinetic energy into internal energy by collision(s), and subsequently the ion dissociates when an excess of vibrational energy is present.4,5 Activation of peptide ions during collisions at kiloelectronvolt energies with neutral targets is often accompanied by substantial losses of kinetic energy by the parent ion (mp).6-9 Since the kinetic energy lost by a parent ion is related to the amount of internal energy converted, its magnitude is a useful quantity. Measurements of energy losses have been used to study (1) Biemann, K.; Martin, S. A. Mass Spectrom. Rev. 1987, 6, 1-76. (2) Carr, S. A.; Hemling, M. E.; Bean, M. F.; Roberts, G. D. Anal. Chem. 1991, 63, 2802-2824. (3) Hunt, D. F.; Yates, J. R., III; Shabanowitz, J.; Winston, S.; Hauer, C. R. Proc. Natl. Acad. Sci. U.S.A. 1986, 83, 6233-6237. (4) Cooks, R. G.; Benyon, J. H.; Caprioli, R. M.; Lester, G. R. Metastable Ions; Elsevier: New York, 1973. (5) McLuckey, S. A. J. Am. Soc. Mass Spectrom. 1992, 3, 599-614. (6) Neumann, G. M.; Derrick, P. J. Org. Mass Spectrom. 1984, 19, 165-170. (7) Boyd, R. K.; Guevremont, R. Rapid Commun. Mass Spectrom. 1988, 2, 1-5. (8) Sheil, M. M.; Derrick, P. J. Org. Mass Spectrom. 1988, 23, 429-435. (9) Bradley, C. D.; Derrick, P. J. Org. Mass Spectrom. 1993, 28, 390-394.
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the dynamics of ion/neutral collisions,10,11 and a direct relationship between mass and energy loss has been noticed.6 Since a sector instrument provides a fixed time scale over which dissociations can be observed, different amounts of internal energy need to be deposited to see products with distinct critical energies of formation. The differences in critical energies should be evident in the kinetic energy lost by the parent ion in the formation of various product ions. Determining the primary sequence of peptides by mass spectrometry requires formation of ions corresponding to successive losses of amino acids. Often, MS/MS spectra of peptides are complicated by the variety of product ion types that correspond to dissociation at different bonds along the backbone of the peptide. The various product ion types are designated by a modified version of the Roepstorff nomenclature,12,13 where charge retention on the N-terminus corresponds to an, bn, and cn ions, and charge retention on the C-terminus corresponds to xn, yn, and zn ions. The existence, for example, of an a-type product ion among a series of y-type product ions can make interpretation of the spectrum difficult. Under high-energy collision conditions (keV), it has been noticed that the type of product ions observed can depend strongly on the structure of the peptide.14,15 On the other hand, spectra resulting from low-energy collision conditions (eV) are dominated by b- and y-type product ions. Thus, highenergy CID spectra can be very ambiguous with respect to the type of ions present. If the type of product ion that each peak corresponds to is known, then interpretation of the spectrum is substantially aided. Since it is generally accepted that the formations of the various types of product ions occur by dissimilar mechanisms,1,3,16-18 then they should have different critical energies. If the various types of product ions have different critical energies, then measuring the kinetic energy lost by the parent ion in the formation of these product ions should reflect this. Thus, we postulate that measuring the energy loss in the formation of product ions may provide a means of identifying product ion types in complex high-energy CID spectra. This (10) Bricker, D. L.; Russel, D. H. J. Am. Chem. Soc. 1986, 108, 6174-6179. (11) Alexander, A. J.; Thibault, P.; Boyd, R. K. J. Am. Chem. Soc. 1990, 112, 2484-2491. (12) Roepstorff, P.; Fohlman, J. J. Biomed. Mass Spectrom. 1984, 11, 601. (13) Johnson, R. S.; Martin, S. A.; Biemann, K.; Stults, J. T.; Watson, J. T. Anal. Chem. 1987, 59, 2621-2625. (14) Biemann, K. Biomed. Environ. Mass Spectrom. 1988, 16, 99-111. (15) Biemann, K. Methods Enzymol. 1990, 193, 455-479. (16) Martin, S. A.; Biemann, K. Int. J. Mass Spectrom. Ion Processes 1987, 78, 213-228. (17) Mueller, D. R.; Eckersley, M.; Richter, W. J. Org. Mass Spectrom. 1988, 23, 217-222. (18) Kenny, P. T. M.; Nomoto, K.; Orlando, R. Rapid Commun. Mass Spectrom. 1992, 6, 95-97. 0003-2700/96/0368-0522$12.00/0
© 1996 American Chemical Society
ability will hinge upon sufficient differences in critical energy to cause noticeable changes in energy loss. Also, the magnitude of energy loss may provide some insight into intramolecular interactions and the mechanism of the dissociation pathway. This may be particularly useful in determining the mechanistic pathway for atypical product ion formations. EXPERIMENTAL SECTION Experiments were performed using a Finnigan MAT 900 instrument which has the forward (EB) geometry. Ions were generated by fast atom bombardment (FAB) using an 8 keV argon beam. Glycerol was used as the liquid matrix with ∼2 µL of a 1 mM peptide solution loaded on a probe tip. The accelerating voltage used in these experiments was 4750 V. Data were obtained by simultaneously scanning the magnetic sector (B) and the electric sector (E) with a constant ratio of B2/ E, which passes product ions formed in the first reaction region (i.e., before the electric sector) to the detector. This means of linked scanning provided the desired energy information of a parent ion when a particular product ion was selected to be transmitted to the detector. The scans were typically 4 s long, and 5-15 scans were summed per spectrum. During the CID experiments, helium (National Welders Supply Co., Charlotte, NC) was used as the collision gas, and the intensity of the parent ion beam was attenuated 65 ( 5%. Under this condition, the peptide ions undergo multiple collisions. Peak centroids were determined by finding the center of the peak at half-height. Since the collision cell was not floated, metastable and CID products overlapped, but the metastable intensities during CID were well under 50% of the CID intensities under the described conditions. Therefore, any metastable intensity in a CID spectrum will not affect the determination of the peak centroid. The peptides leucine enkephalin, des-Arg1-bradykinin, bradykinin, and substance P were purchased from Sigma Chemical Co. (St. Louis, MO). The leucine enkephalin variants were synthesized in-house. Gly-Pro-Gly-Gly was obtained from Mann Research Laboratories (New York, NY). To guarantee the accuracy of the kinetic energy lost during CID for these initial studies, two experiments were performed. The kinetic energy of the ions in the absence of collision gas was determined to serve as a reference. This was accomplished by observing the metastable dissociation of a parent ion to a particular product ion. The kinetic energy lost was then determined by subtracting the kinetic energy of the product ion after CID from the kinetic energy of the product ion in the absence of collision gas (metastable ion). It should be emphasized that for this initial study, energy losses were measured only for product ions which can be observed in the CID and metastable spectra. Requiring metastable dissociations precluded the measurement of some product ion types in this study (e.g., d) but was considered necessary to maximize the accuracy of the measurements. Future studies will undertake energy loss measurements of other product ion types once a better method to determine the energy loss without a metastable reference has been implemented on our instrument. Since the data system of our instrument plotted the results of a scan on a mass scale, conversion to an energy scale was needed. The kinetic energy of the product ion (KE) could be obtained from the spectrum by taking the ratio of the product ion mass (md) to the parent ion mass (mp) from a parent scan (B2/E) and multiplying this value by the accelerating voltage (V):
KE ) (md/mp)V
(1)
Calculating KE from a spectrum corresponding to the CID experiment and subtracting it from the value obtained during the metastable experiment gives the kinetic energy lost by the product ion (∆Ed):
∆Ed ) KE(m*) - KE(CID) ∆Ed ) (mdm*/mpm*)V - (mdCID/mpCID)V
(2)
The experimentally observed ∆Ed values were converted to the corresponding energy losses (∆Ep) of the parent ions by the simple relation,
∆Ep ) (mp/md)∆Ed
(3)
THEORY Variations in energy losses can be qualitatively explained by looking at the Rice-Rampsberger-Kassel (RRK) expression,
( )
k)ν
- 0
N-1
(4)
where k is the rate of dissociation, ν is the frequency factor, is the internal energy of the ion, 0 is the critical energy for the dissociation, and N is the number of degrees of freedom. In our experiments, since k has a fixed range and, for a given parent ion, N is fixed, changes in 0 will require changes in , assuming frequency factors are similar. It is proposed that the kinetic energy lost by the parent ion is proportional to and that differences in kinetic energy loss reflect different 0 and, therefore, different dissociation mechanisms. There are two assumptions implicit in this proposed relationship. First, the overall internal energy of the parent ion is the sum of the energy converted from the collision and the internal energy of the ion resulting from the desorption/ionization event. Thus, it is assumed that internal energy from the desorption/ionization process is a constant and does not affect the dissociation pathway. The second assumption is that other energy loss processes (e.g., gain in kinetic energy of the He collision partner) are also constant and independent of the dissociation pathway. Both these assumptions should be valid if the collisional activation process is independent of the dissociation, as is commonly accepted.5,19 It should be noted here that the magnitude of the energy losses will depend on the initial kinetic energy of the parent ion8 and the internal energy gained by the parent ion during the ionization process (e.g., by different ionization techniques).6,7 While it is well known that the RRK equation is not a completely accurate description of ion dissociation, it has long been realized that the theory can give reasonable results if the number of vibrational modes (N) is arbitrarily lowered by a factor of 3-5 from its defined value.4,20-23 If eq 4 is rearranged to give internal energy () as a function of the number of degrees of (19) Cooks, R. G. Collisional Spectroscopy; Plenum Press: New York, 1979. (20) Chupka, W. A. J. Chem. Phys. 1959, 30, 191. (21) Kropf, A.; Eyring, E. M.; Wahrhaftig, A. L.; Eyring, H. J. Chem. Phys. 1960, 32, 149. (22) Stemer, B.; Giese, C. F.; Inghram, M. G. J. Chem. Phys. 1961, 34, 189.
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523
Figure 1. Theoretical plot of internal energy versus the number of degrees of freedom from eq 5 at critical energies of 1, 2, and 3 eV.
Figure 3. (a) Parent scan of the a4 ion of YGRFL, metastable dissociation. (b) Parent scan of the a4 ion of YGRFL, collision-induced dissociation (CID). The energy loss is evident in the apparent shift to higher mass. Table 1. Compilation of Energy Losses Associated with the Formation of Various Product Ions from a Number of Different Peptides energy loss values (eV) Figure 2. Theoretical plot of internal energy versus the number of degrees of freedom from eq 5 at a critical energy of 1 eV and varying frequency factors (a, 1 × 1010; b, 1 × 1011; c, 1 × 1012; d, 1 × 1013).
freedom (N), eq 5 results. It can be seen from Figure 1 that the
)
-0 e
[ln(k/ν)]/(N-1)
-1
(5)
an peptide
RESULTS AND DISCUSSION Figure 3 illustrates a typical result obtained in these experiments. The metastable dissociation (a) and the dissociation after (23) Larsen, K. Fundamental Aspects of Organic Mass Spectrometry; Verlag Chemie: Weinheim, 1978.
524 Analytical Chemistry, Vol. 68, No. 3, February 1, 1996
MW n ∆E n ∆E
G-P-G-G
286
G-F-L leucine enkephalin
335 2 555 4
2 3 5.0 2 5.7 3 4
3.3 2.8 3.1 7.4 3.5
Y-G-R-F-L
654 3 11.0 3 4 13.0 4 903 7 21.6
7.0 5.9
des-Arg1-bradykinin
plot of eq 5 is approximately a straight line. Figure 1 is a generic plot where k ) 1 × 106 and ν ) 1 × 1013. The three lines correspond to arbitrarily chosen critical energies of 1, 2, and 3 eV. As will be demonstrated later, the experimental data follow this same linear trend, and so it seems that the eq 4 is adequate for making qualitative explanations. The assumption made above that the frequency factors (ν) need to be similar is a very loose criterion. In fact, a 3 order of magnitude change in the frequency factor results in less of a change in the internal energy requirements than does a 1 eV change in the critical energy (0). Figure 2 shows the effect of changing the frequency factor at a given critical energy.
bn
bradykinin substance P
1060 5 6 8 1347 7
yn
other
n
∆E
3
2.2
2 2 3 4 3 4 7
type
2.0 4.4 3.6 3.7 4.9 b4 + OH 8.8 z3 7.0 b7 + OH z4 7 11.1 b8 + OH
22.9 6 12.8 19.7 8 17.8 15.1 23.6 10 11.1
∆E
3.0 7.2 5.4 9.6 5.6
collisional activation (b) of YGRFL in the formation of the a4 ion show the energy loss after CID. In the CID case (b), the energy loss is evident in the apparent shift to higher masses (lower kinetic energy) in the spectrum. By comparing these two spectra and performing the calculations described in the Experimental Section, the energy loss was determined to be 13.0 eV. Table 1 shows a compilation of some energy losses associated with different product ion types for a number of peptides. These values are plotted with respect to parent ion mass in Figure 4, which allows the trends to be easily seen. The standard deviations of the measurements are between 10 and 20% of the indicated values for the majority of the ions. As can be seen, in many cases product ions of the same type have similar energy losses for a given parent
Figure 5. Proton bridge between nitrogen on the side chain of arginine and the carbonyl oxygen on the adjacent amino acid.
Figure 4. Plot of energy loss values versus parent ion mass for a-, b-, and y-type product ions (+, a-type; 4, b-type; O, y-type). The values for a given fragment type have been averaged for each peptide, but the anomalous values are not included in the average.
ion. There are, however, exceptions for which possible explanations will be provided (vide infra). Also note that N-terminal aand b-type product ions show greater energy losses, as a general rule, than the C-terminal y-type product ions. Likewise, a-type product ions show greater energy losses than b-type product ions. These results make sense in view of the accepted mechanisms for the formation of these type product ions. Data from spectra of peptides under low-energy CID conditions show that b- and y-type product ions dominate the fragmentation patterns, presumably due to their lower energies of formation.24,25 The lower energy loss magnitudes for the b- and y-type product ions with respect to the a-type product ions agree with the notion that band y-type product ions result from lower internal energy deposition during collisional activation than a-type product ions. Not enough measurements of z-type product ions were made to allow for a significant plot of these values; however, the two measurements obtained for the z-type product ions provide some information about the formation of this type product ion. In fact, a number of different product ion types are observed in CID spectra, but the their lack of intensity in the metastable spectra prevents an accurate measurement of the energy losses by the method described in the Experimental Section. The magnitude of the energy losses for the z-type product ions of des-Arg1bradykinin and YGRFL fall in between the values for a- and b-type product ions. This result and the fact that z-type product ions are predominately found in high-energy spectra13 suggest that its formation is a higher energy process than the formation of either the b- or y-type product ions. A general trend that is seen in all cases is that the energy loss for a particular type of product ion increases with mass (Figure 4), as had been noted before.6 As the mass of the peptide increases, the number of degrees of freedom (N) is also increasing. With increased degrees of freedom, there are more vibrational modes to distribute the internal energy among; therefore, more energy is required for dissociation. This experimental plot follows the same trend seen in the theoretical plot of Figure 1. The correlation coefficients for the lines in Figure 4 of the a-, b-, (24) Bean, M. F.; Carr, S. A.; Thorne, G. C.; Reilley, M. H.; Gaskell, S. J. Anal. Chem. 1991, 63, 1473-1481. (25) Alexander, A. J.; Thibault, P.; Boyd, R. K.; Curtis, J. M.; Rinehart, K. L. Int. J. Mass Spectrom. Ion Processes 1990, 98, 107-134.
and y-type product ions are 0.999, 0.990, and 0.970, respectively. In this graph, the average energy loss values for a given fragment type are plotted for each peptide, but the values that are considered exceptions (vide infra) are not included in the average. A product ion that shows a particularly interesting result is the (b8 + OH) ion of bradykinin (RPPGFSPFR). Previous studies using isotopic labeling have shown that this ion results from the loss of the C-terminal arginine and not the N-terminal arginine, which would give the isomeric y8 ion.26 The proposed mechanism for the formation of this ion is a rearrangement involving the transfer of a hydrogen to the carbonyl oxygen on the amino acid phenylalanine (F), retention of the carboxyl oxygen, and subsequent losses of -CO and -NHdCH-Arg. An alternate but similar mechanism has recently been advanced.27 Ion decompositions that involve rearrangements generally have lower critical energies than simple cleavages, and in fact, it is seen that the energy loss for the (b8 + OH) ion is substantially lower than that for other product ions of bradykinin. The (b4 + OH) ion of YGRFL and the (b7 + OH) of des-Arg1-bradykinin also show substantially lower energy losses than the other product ions of those peptides, suggesting that a similar rearrangement is involved in their dissociation. For an unknown peptide, such anomalously low energy losses may be an indication of some type of rearrangement occurring in the formation of a product ion. A common feature of the peptides discussed above is the presence of arginine. Another unique trend in the energy losses can be noticed in the peptides that contain arginine. Product ions that correspond to dissociations of a bond on the amino acid adjacent to the arginine tend to show unusually large energy losses. This trend can be seen in the b8 ion of bradykinin (RPPGFSPFR), the y4 and a4 ions of YGRFL, and the a7 ion of des-Arg1-bradykinin (PPGFSPFR). These peptides can presumably adopt a conformation where the side chain of the arginine is close to the carbonyl oxygen on the adjacent amino acid. If the proton is assumed to be localized on the side chain of arginine, it can form a proton bridge between the carbonyl oxygen and a nitrogen on the arginine side chain (Figure 5). More energy, then, is needed to disrupt this interaction and yield the resulting product ion. A more detailed discussion of this interaction has been presented elsewhere.27 CONCLUSIONS The amount of kinetic energy lost by a particular peptide ion during CID is related to the type of dissociation that occurs. (26) Thorne, G. G.; Ballard, K. D.; Gaskell, S. J. J. Am. Soc. Mass Spectrom. 1990, 1, 249-257. (27) Vachet, R. W.; Asam, M. R.; Glish, G. L. Submitted to J. Am. Chem. Soc.
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These variations in energy loss are attributed to the differences in critical energy and mechanisms of formation of the various types of product ions. The product ion type can be classified by measuring the energy loss of a parent ion in the formation of a particular product ion and comparing this value with values predicted from the energy loss versus mass relationships depicted in Figure 2. While energy loss measurement may not be feasible in cases with a very limited amount of sample, if sufficient sample is available, such measurements will simplify determination of the peptide sequence from the MS/MS spectrum. Energy loss measurements may be particularly useful in identifying unex-
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pected dissociations, such as formation of (bn + OH)+, which may otherwise be mistakenly identified as another product ion type. ACKNOWLEDGMENT This work was supported by NIH GM49852. Received for review September 5, 1995. November 7, 1995.X
Accepted
AC950893R X
Abstract published in Advance ACS Abstracts, December 15, 1995.
