RAPID COMMUNICATIONS IN MASS SPECTROMETRY, VOL. 6,658-662 (1992)
Charge Promotion of Low-energy Fragmentations of Peptide Ions Odile Burlet,+ Ralph S. Orkiszewski, Kevin D. Ballard and Simon J. Gaskell* Center for Experimental Therapeutics, Baylor College of Medicine, Houston, TX 77030, USA SPONSOR REFEREE: Professor M. J. Bertrand, UniversitC de Montreal, Montreal, Quebec, Canada
We have examined the hypothesis that structural features which predispose to localization of charge at a strongly favored site are not conducive to the low-energy fragmentation of peptide ions via a multiplicity of pathways. Consistent with this proposal, it is demonstrated that the formation of N- or C-terminal pre-charged derivatives is detrimental to the formation of sequence-specific product ions following low-energy collisional activation. Protonation of pre-charged derivatives (yielding doubly charged ions) restores favorable fragmentation properties; the effect is attributed to the fragmentation-directingproperties of the proton which may occupy one of several sites. Similarly, a doubly protonated peptide which incorporates a C-terminal arginine residue as a single strongly favored site of protonation exhibits favored low-energy fragmentations attributable to location of the second proton at one of several sites remote from the C-terminus.
There is continuing debate over the relative merits of low- and high-energy collisional activation in tandem mass spectrometric analyses of peptides and other biopolymers. Extensive studies of high-energy collisionally activated dissociation (CAD) using four-sector instruments'-6 have established the major fragmentation types. These include single cleavages of the peptide backbone with charge retention on the N- or C-terminal fragment, depending on amino acid composition. In addition, concomitant sidechain cleavages occur. The pioneering work of Hunt and his colleague^,^ together with more recent comparisons of hybrid and four-sector instruments'-'' have indicated that the decompositions of protonated peptides can be substantially more complex in the low-energy CAD regime compared with the higk-energy experiment. In addition to several series of ions arising from single peptide chain cleavages, low-energy CAD product-ion spectra may include prominent 'internal fragment^'^. (which are less frequently significant in the high-energy CAD spectra"), together with the products of ion rearrangements.l2 The relationship between amino acid sequence and low-energy decompositions of protonated peptides remains imperfectly understood. In recent workI3 we described marked differences in low-energy CAD between protonated peptides incorporating C-terminal arginine and mid-chain cysteine residues, and analogues in which the cysteine had been replaced by cysteic acid. The product-ion spectra of the cysteic acid analogues exhibited a strong promotion of the formation of multiple members of the series of sequence ions corresponding to peptide bond cleavage with charge retention on the C-terminus. Removal of the arginine residue abolished the effect. The observations were attributed to an intra-ionic interaction between the cysteic acid and arginine sidechains, resulting in a reduced propensity for proton location on the arginine residue. It was proposed13 that these findings were consistent with the general hypothesis that an 'Also at the Department of Chemistry, University of Houston, Houston, TX 77204-5641, USA. * Author to whom correspondence should be addressed.
0951-4 1Y8/Y2/1 10658-05 $07S O @ 1992 by John Wiley & Sons, Ltd.
increased heterogeneity (with respect to site of charge) of the protonated peptide precursor population is beneficial to the yield of product ions via multiple, chargedirected, low-energy fragmentation pathways. A variety of evidence from the literature is consistent with this hypothesis. Poulter and Taylor,8 for example, have suggested that, in general, the presence of a terminal arginine residue is detrimental to the observation of sequence ions following low-energy CAD of protonated peptides. Comparative studies" of protonated substance P (RPKPQQFFGLM-NH,) and des-Arg'-substance P have indicated a substantially higher yield of structurally informative fragment ions following low-energy CAD of the truncated peptide ion that lacks the arginine residue. In contrast, high-energy CAD yielded superior results for protonated substance P. lo Thus, previous studies suggest that structural features which predispose to localization of charge at a strongly favored site are not conducive to the lowenergy fragmentation of peptide ions via a multiplicity of pathways. The consequence for the effective tandem mass spectrometric characterization of peptides using low-energy CAD is that sequence information may be incomplete. If this argument is correct, then the following specific hypotheses may be proposed: (i) formation of pre-charged derivatives will be detrimental to effective peptide analysis by low-energy CAD. The fixed site of charge will promote only proximal fragmentations, and structural detail remote from the site of charge will not be apparent from the product-ion spectrum. The preparation of pre-charged derivatives of peptides has been advocated as a means of improving ion yield following fast-atom bombardment' and of favoring the detection of particular fragments (retaining the N- or C-terminus, depending on the site of derivatization) following high-energy CAD.^.^. I4-l7 For example, Biemann and coworkers have shown that d-series ions (corresponding to concomitant peptide bond and sidechain cleavages, with charge retention on the N-terminal fragment) are favored during high-energy CAD of peptide ions incorporating a pre-charged N-terminal derivative group.I6 Formation of these ions Received 4 September 1992 Accepted 4 September I992
CHARGE PROMOTION OF LOW ENERGY FRAGMENTATIONS
necessarily occurs remote to the site of charge; it is generally considered that such charge-remote fragmentations have a relatively high energy requiremenP and are therefore not expected as products of low-energy decompositions. Low-energy CAD product-ion spectra of pre-charged derivatives of peptides are therefore predicted to reflect poor yields of sequence-specific fragment ions. (ii) Incorporation of a second charge into ions in which the first charge is fixed (either by a strongly favored site for protonation or by a precharged derivative group) will promote additional diagnostic fragmentation by providing additional possibilities of charge-proximal cleavages. The present report describes analyses designed to assess these specific hypotheses.
659
n
on RI
R,
I ch=c=N-o I'
Pre-charged N-terminal derivative
Pre-charged C-terminal derivative
Scheme 1
EXPERIMENTAL Materials. All peptides are designated using the standard single letter codes for the amino acid residues. GFLCGHYR was provided by D r R. Cook (Baylor College of Medicine, Houston, TX, USA). SIGSLAK and FVQWLMNT (glucagon, 22-29) were purchased from Sigma (St Louis, MO, USA) and Bachem (Torrance, CA, USA), respectively. N- Terminal derivatization of peptides. The procedure adopted was that described by Wagner et aI.l4 Bromoethyl-triphenylphosphonium bromide was prepared by addition of triphenylphosphine (220 mg) to dibromoethane (1 mL). The mixture was stirred at 80 "C for 22 h. The precipitate was washed three times with diethyl ether, dried and stored under nitrogen. The lyophilized peptide (100-300 nmol) was dissolved in 0.16 M potassium borate buffer (pH 9.0; 1mL). To the peptide solution was added a 20-fold molar excess of bromoethyl-triphenylphosphonium bromide dissolved in acetonitrile. The solution was vortex-mixed and allowed to react at 37 "C for 3 h. The reaction mixture was loaded on a Sep-Pak octadecylsilyl silica cartridge (Waters Division of Millipore, Milford, MA, USA). The cartridge was eluted with water (5 mL) and acetonitrile water ( l / l ; 10 mL); the latter fraction, containing the peptide derivative, was dried for subsequent mass spectrometric analysis. C-Terminal deriuatization of peptides. The procedure of Wagner et aI.l4 was used. Aminoethyl-triphenylphosphonium bromide was prepared by solution of bromotriphenylphosphonium bromide (50 mg) in absolute ethanol (250pL) and dropwise addition of ammonium hydroxide (70 pL). The mixture was stirred for 1 h at 20°C and evaporated under a stream of nitrogen. The lyophilized peptide (100-300 nmol) was dissolved in a dilute aqueous solution of trifluoroacetic acid (pH 5.0) and a 20-fold molar excess of dicyclohexylcarbodiimide was added to the solution. The solution was vortex-mixed and heated at 37 "C for 5 min. A 20fold molar excess (over the peptide) of aminoethyltriphenylphosphonium bromide was added to the solution and reaction was allowed to proceed for 3 h at 37 "C. The reaction mixture was lyophilized. Mass spectrometry. All analyses were peformed using a ZAB SEQ hybrid mass spectrometer (VG Analytical, Manchester, UK) with the configuration, BEqQ (where B = magnetic sector, E = electric sector, q = RF-only quadrupole collison region, Q =
+
quadrupole mass filter). Ionization by fast-atom bombardment (FAB) used xenon atoms with energies of 8 k e V as the primary beam. The matrix was a 1 / 1 mixture of thioglycerol and 2,2'-dithiodiethanol, saturated with oxalic acid. Low-energy collisional activation (in q) used argon as collision gas at an estimated pressure (in the collision region) of 1.8 - 3.4 X mBar, achieving 50-60% attenuation of the precursor ion beam. The collision energy was 10-15 eV. Product-ion spectra were acquired via the VG 11-250 data system in the 'multichannel analyzer' mode. The nomenclature used for the peptide fragment ions is that suggested by Roepstorff and F ~ h l m a n , extended '~ (as described in the Results and Discussion section) to the description of the products of pre-charged derivatives. RESULTS AND DISCUSSION The role of the charge site in determining the extent and pathways of low-energy decompositions of peptides has been examined by comparison of the production spectra of protonated peptides and of pre-charged derivatives. We have used the derivatization procedures described by Wagner et al. 14. 's to permit the introduction of a charged functional group (the ethyltriphenylphosphonium moiety) at either the C- or the N-terminus (Scheme 1). Figure 1 shows a comparison of the low-energy CAD product-ion spectra of protonated FVQWLMNT (glucagon 22-29) and the N-terminal ethyl-triphenylphosphonium derivative. The protonated peptide yields prominent B-series sequence ions, accompanied by Y-series ions of lower abundance. The favorable low-energy fragmentation properties of this protonated peptide are interpreted to reflect the lack of a strongly favored site of protonation, with a consequently heterogeneous (with respect to site of charge) precursor-ion population. The product-ion spectrum of the pre-charged derivative (Fig. l(b)) is dominated by the CH2=CH-P+(C6H5)3 fragment; peptide sequence ions ('A- and 'B-series) are observed with very low signal-to-noise ratios. (Although the nomenclature used for the peptide fragments is based on the recommendations of Roepstorff and Fohlman, for the pre-charged derivatives, hydrogen additions or deletions from the fragment ions are shown relative to the pre-charged precursor; the equivalent nomenclature for the protonated . peptides is conventionally shown relative to the neutral molecule.) Thus, prep-
660
CHARGE PROMOTION O F LOW ENERGY FRAGMENTATIONS 1038
M+ 1326
Figure 1. Product-ion spectra recorded following low-energy CAD of precursors derived from the octapeptide, FVQWLMNT. (a) The protonated peptide, [M + HI', mlz 1038. (b) The pre-charged N-terminal ethyl-triphenylphosphonium derivative, M', m / z 1326.
aration of a pre-charged derivative is detrimental to the abundances of structurally diagnostic fragment ions. Figure 2(a) and (b) shows the spectra obtained by low-energy CAD of the protonated heptapeptide, SIGSLAK, and of the C-terminal ethyl-triphenylphosphonium derivative of the corresponding amide, respectively. The spectrum of the protonated peptide contains extensive sequence information; all members of the Y-series are present and B2-B, ions are also observed. In addition, prominent ions corresponding to loss of water from B-series ions are present. The diverse fragmentations of this protonated peptide are again attributed to the lack of a strongly favored site of protonation, resulting in a heterogeneous (with respect to site of charge) precursor-ion population. The slightly greater prominence of fragment ions retaining the C-terminus (though this is not pronounced) is taken to result from the stabilization of product ions which include the somewhat basic lysine residue. The product-ion spectrum (Fig. 2( b)) of the pre-charged derivative (now located at the C-terminus) is again dominated by a fragment, CH2=CH-P+(C6H5)3, associated with the derivative group. No sequence-related product ion exceeds 2.5% of the relative abundance of this fragment; Yi - Yi ions are, however, detectable. The yields of sequence-specific product ions from SIGSLAK [M + H]+ and from the SIGSLAK precharged derivative M + ions were compared by recording spectra obtained with similar precursor-ion abundances. This comparison indicated, for example, that the Y; ions derived from the native peptide were detected with a signal-to-noise ratio approximately six times that observed for the Y; ion derived from the pre-charged derivative. The observation (albeit at very low abundance) of the products of apparent charge-remote processes in the spectra of the pre-charged derivatives (Y'-series
ions in the case of thue C-terminal derivative, and principally 'A-series ions for the example of the N-terminal derivative) may have two explanations. (i) The peptide sequence ions may indeed result from
IM+W Y5" 475
675
I
Figure 2. Product-ion spectra recorded following low-energy CAD of precursors derived from the heptapeptide, SIGSLAK. (a) The protonated peptide, [M+H]+, mlz 675. (b) The pre-charged C-terminal ethyl-triphenylphosphonium derivative of the amide analogue, M+, m / z 962. (c) The protonated, pre-charged derivative, [M+H]'+, mlz4$1.5. m l z ratios are rounded down to the nearest integral or half-integral value.
CHARGE PROMOTlON OF LOW ENERGY FRAGMENTATIONS
66 1
Figure 3. Product-ion spectra recorded following low-energy CAD of precursor derived from the octapeptide, GFLCGHYR. (a) The protonated peptide, [M+ HI+, mlz 952. (b) The doubly protonated peptide, [M+2H12+, mlz476.5. mlz Ratios are rounded down to the nearest integral or half-integral value. The assignment of mlz 318 as the doubly charged ion, Y'y, rather than thc singly charged B, (which would have a similar m / z ratio), is preferred on the basis of the peak width apparent in the expanded spectrum.
charge-remote fragmentation of a very minor subpopulation of precursor ions of sufficiently high internal energy. Wagner et ~ 1 . 'l5 ~ have . previously noted the presence of 'A ions in the high-energy C A D production spectra of N-terminal ethyl-triphenylphosphonium derivatives of peptides; the equivalent C-terminal derivatives, however, yielded 'Y product ions following high-energy CAD. Thus, there are mechanistic differences, yet to be elucidated, between the two decomposition regimes. (ii) The peptide sequence ions may be associated with charge proximal processes resulting from the adoption of the appropriate conformation of the gas-phase precursor ion. (Previous studies have highlighted the significance of gas-phase conformation in determining low-energy fragmentations of peptides.'*. 2") The most striking feature of the spectra of the precharged derivatives, however, is the very low relative abundances of sequence-specific, low-energy fragmentations, consistent with the predicted detrimental effect of the fixed site of charge. These data are therefore in accord with our initial hypothesis that multiple lowenergy decompositions of peptides are promoted by a heterogeneous (with respect to site of charge) population of precursor ions, with the corollary that a fixed site of charge affords unfavorable fragmentation properties. Extending this same logic suggests that protonation of a pre-charged peptide derivative (yielding a singly protonated, but doubly charged, ion) would restore the propensity to fragment via multiple pathways. Figure 2(c) shows the product-ion spectrum of the protonated, pre-charged derivative of SIGSLAK. The C H d H - P + ( C , H , ) , fragment ion associated with the pre-charged derivative group now appears with low relative abundance (in contrast to the spec-
trum of the singly charged derivative, Fig. 2(b)) and peptide sequence ions (both C- and N-terminal) assume much greater prominence. In particular, fragment ions resulting from cleavage between the isoleucine and glycine residues (yielding Y;, Y!;, and B2 (and the associated A2 product ion)) are prominent, suggesting that Coulombic repulsion is effective in favoring a site of protonation which promotes these cleavages via charge proximal mechanisms. The failure to observe B , , YA or Yl fragment ions, however, which would be predicted to result from protonation of the serine/ isoleucine peptide bond, suggests mechanistic complexities which require further investigation. In addition, the absence of an ion of m / z 674, complementary to the derivative-related ion of m / z 289, may indicate a significant contribution from sequential fragmentation processes*' which might be probed by tandem mass spectrometric (MS/MS/MS) techniques. The same principles apply to a comparison of the product-ion spectra obtained by low-energy C A D of singly and doubly protonated GFLCGHYR (Fig. 3). The spectrum of the singly protonated cysteinecontaining analogue is shown in Fig. 3(a). The spectrum is dominated by the Yy ion, reflecting the heavily favored charge location at the strongly basic arginine residue (though the presence of other sequence ions of low relative abundance may suggest minor proportions of the precursor population which incorporate protonation at alternative sites). As previously reported, superior fragmentation properties were observed for the protonated analogue in which the cysteine residue is oxidized to cysteic acid; the effect was attributed to a reduced propensity for charge localization on the arginine residue resulting from interaction with the cysteic acid sidechain. The FAB mass spectrum of the
662
CHARGE PROMOTION O F LOW ENERGY FRAGMENTATIONS
cysteine-containing peptide (GFLCGHYR) includes a weak signal corresponding to the doubly protonated molecule. Despite its low abundance, selection of the doubly protonated precursor during tandem mass spectrometric analysis is facilitated by its appearance at approximately half-integral mass. The product-ion spectrum (Fig. 3(b)) shows prominent cleavages occurring remote from the arginine residue, consistent with the notion that charge repulsion favors a second site of protonation distant from the presumed most favored site (the arginine residue). Thus, cleavages of the phenylalanine/leucine bond (yielding B2 and Y;, Y 0’) and the leucine/cysteine bond (yielding Yy, YI;’) are strongly promoted. (The possible fragment ions, B3and Y r;r, both arising from cleavage of the leucine/cysteine bond, have similar mlz ratios. The assignment of rnlz 318 to Y’; (that is, a doubly charged ion) is tentatively preferred based on the peak width apparent from the expanded spectrum (not shown). The relative contributions of B, and Yy’fragments to the signal at mlz 318 do not, however, affect the validity of the argument that cleavage of the leucine/cysteine bond is promoted by proximal protonation.) Interestingly, fragmentation of the peptide bond between residues 1 and 2 (glycine and phenylalanine) is not observed for the doubly protonated peptide (Fig. 3(b)), an observation that parallels that made for the protoanted, pre-charged derivative of SIGSLAK (Fig. 2(c)). The tandem mass spectrometric analysis of peptides containing a C-terminal arginine residue is a common requirement in protein characterization because of the frequent observation of such peptides in mixtures derived from protein hydrolyses using the enzyme, trypsin. Electrospray-tandem mass spectrometry, involving the low-energy C A D of doubly protonated precursors, has been particularly effective for the analysis of tryptic digest^.'^-^' This success may be attributed, in part, to the promotion of peptide ion fragmentation by the second proton, the first being strongly associated with the arginine residue. CONCLUSION The evidence of the unfavorable low-energy fragmentation properties of pre-charged derivatives of peptides reinforces the view that the promotion of multiple diagnostic fragmentations by low-energy CAD of peptide ions is contingent upon a precursor-ion population that is heterogeneous with respect to the location of charge. Equivalently, native or introduced structural features which lead to a single preponderant site of charge are detrimental to the generation of informative product-ion spectra under low-energy CAD conditions. When protonation of a peptide takes place at a single
strongly favored site (such as the C-terminal arginine residue frequently observed for peptides derived by tryptic hydrolysis of proteins), more favorable fragmentation properties may be observed for the doubly protonated analogue.
REFERENCES 1. K. Biemann and S. A. Martin, Mass Specfrom. Reo. 6 , 1 (1990). 2. R. S. Johnson, S. A. Martin and K. Biemann, Inf. J. Mass Specfrom. Ion Processes 86, 137 (1988). 3. K. Biemann, Biomed. Enoiron. Mass Spectrom. 16, 99 (1988). 4. K. Biemann, Methodr Enzymol. 193, 455 (1990). 5. S. Kaur, K. F. Medzihradszky, Z. Yu, M. A. Baldwin, B. L. Gillece-Castro, F. C. Walls, B. W. Gibson and A. L. Burlingame, in Biological Mass Spectrometry, pp. 285-309, ed. by A. L. Burlingame and J. A. McCloskey, Elsevier, Amsterdam (1990). 6. J. T. Stults, in Methods in Biochemical Analysis, Vol. 34: Biomedical Applications of Mass Spectromefry, pp. 145-201, ed. by C. H. Suelter and J. T. Watson, Wiley, New York (1990). 7. D. F. Hunt, J. R. Yates, 111, J. Shabanowitz, S. Winston and C. R. Hauer, Proc. Natl Acad. Sci. USA 83, 6233 (1986). 8. L. Poulter and L. C. E. Taylor, Inf. J . Mass Spectrom. Ion Processes 91, 183 (1 989). 9. A. J. Alexander, P. Thibault, R. K. Boyd, J. M. Curtis and K. L. Rinehart, Inf. J. Mass Spectrom. Ion Processes 98, 107 (1990). 10. M. F. Bean, S. A. Carr, G . C. Thorne, M. H. Reilly and S. J. Gaskell, Anal. Chem. 63, 1473 (1991). 11. K. D. Ballard and S. J. Gaskell, Inf. J . Mass Spectrom. Ion Processes 111, 173 (1991). 12. G. C. Thorne, K. D. Ballard and S. J. Gaskell, J. A m . SOC.Mass Spectrom. 1, 249 (1990). 13. 0. Burlet, C.-Y. Yang and S. J. Gaskell, J. Am. SOC. Mass Spectrom. 3, 337 (1992). 14. D. S. Wagner, A. Salari, D. A. Gage, J. Leykam, J. Fetter, R. Hollingsworth and J . T. Watson, Biol. Mass Spectrom. 20, 419 (1991). 15. J. T. Watson, D. S. Wagner, Y.-S. Chang, J . Strahler, S. Hanash and D. A. Gage, Int. J. Mass Specfrom. Ion Processes 111, 191 (1991). 16. J. E. Vath and K. Biemann, Inf.J . Mass Spectrom. Ion Processes 100, 287 (1990). 17. J. T. Stults, S. McCune and J. Lai, Proceedings of fhe 39th Conference on Mass Specfrometry and Allied Topics, Nashville, TN, p. 765, ASMS, East Lansing (1991). 18. J . A d a m and M. L. Gross, J . A m . Chem. Soc. 108,6915 (1986). 19. P. Roepstorff and J. Fohlman, Biomed Mass Spectrom. 11, 601 (1984). 20. K. D. Ballard and S. J. Gaskell, J. A m . Chem. SOC. 114, 64 (1992). 21. X.-J. Tang and R. K. Boyd, Rapid Commun. Mass Spectrom. 6 , 651 (1992). 22. T. R. Covey, E. C. Huang and J . D. Henion, Anal. Chem. 63, 1193 (1991). 23. C. G . Edmonds and R. D. Smith, Methods Enzymol. 193, 412 (1990). 24. P. Thibault, S. Pleasance, M. V. Laycock, R. M. MacKay and R. K. Boyd, Int. J. Mass Spectrom. Ion Processes 111, 317 (1991). 25. I. Jardine, Methods Enzymol. 193, 441 (1990).
Charge Promotion of Low-energy Fragmentations of Peptide Ions Odile Burlet,+ Ralph S. Orkiszewski, Kevin D. Ballard and Simon J. Gaskell* Center for Experimental Therapeutics, Baylor College of Medicine, Houston, TX 77030, USA SPONSOR REFEREE: Professor M. J. Bertrand, UniversitC de Montreal, Montreal, Quebec, Canada
We have examined the hypothesis that structural features which predispose to localization of charge at a strongly favored site are not conducive to the low-energy fragmentation of peptide ions via a multiplicity of pathways. Consistent with this proposal, it is demonstrated that the formation of N- or C-terminal pre-charged derivatives is detrimental to the formation of sequence-specific product ions following low-energy collisional activation. Protonation of pre-charged derivatives (yielding doubly charged ions) restores favorable fragmentation properties; the effect is attributed to the fragmentation-directingproperties of the proton which may occupy one of several sites. Similarly, a doubly protonated peptide which incorporates a C-terminal arginine residue as a single strongly favored site of protonation exhibits favored low-energy fragmentations attributable to location of the second proton at one of several sites remote from the C-terminus.
There is continuing debate over the relative merits of low- and high-energy collisional activation in tandem mass spectrometric analyses of peptides and other biopolymers. Extensive studies of high-energy collisionally activated dissociation (CAD) using four-sector instruments'-6 have established the major fragmentation types. These include single cleavages of the peptide backbone with charge retention on the N- or C-terminal fragment, depending on amino acid composition. In addition, concomitant sidechain cleavages occur. The pioneering work of Hunt and his colleague^,^ together with more recent comparisons of hybrid and four-sector instruments'-'' have indicated that the decompositions of protonated peptides can be substantially more complex in the low-energy CAD regime compared with the higk-energy experiment. In addition to several series of ions arising from single peptide chain cleavages, low-energy CAD product-ion spectra may include prominent 'internal fragment^'^. (which are less frequently significant in the high-energy CAD spectra"), together with the products of ion rearrangements.l2 The relationship between amino acid sequence and low-energy decompositions of protonated peptides remains imperfectly understood. In recent workI3 we described marked differences in low-energy CAD between protonated peptides incorporating C-terminal arginine and mid-chain cysteine residues, and analogues in which the cysteine had been replaced by cysteic acid. The product-ion spectra of the cysteic acid analogues exhibited a strong promotion of the formation of multiple members of the series of sequence ions corresponding to peptide bond cleavage with charge retention on the C-terminus. Removal of the arginine residue abolished the effect. The observations were attributed to an intra-ionic interaction between the cysteic acid and arginine sidechains, resulting in a reduced propensity for proton location on the arginine residue. It was proposed13 that these findings were consistent with the general hypothesis that an 'Also at the Department of Chemistry, University of Houston, Houston, TX 77204-5641, USA. * Author to whom correspondence should be addressed.
0951-4 1Y8/Y2/1 10658-05 $07S O @ 1992 by John Wiley & Sons, Ltd.
increased heterogeneity (with respect to site of charge) of the protonated peptide precursor population is beneficial to the yield of product ions via multiple, chargedirected, low-energy fragmentation pathways. A variety of evidence from the literature is consistent with this hypothesis. Poulter and Taylor,8 for example, have suggested that, in general, the presence of a terminal arginine residue is detrimental to the observation of sequence ions following low-energy CAD of protonated peptides. Comparative studies" of protonated substance P (RPKPQQFFGLM-NH,) and des-Arg'-substance P have indicated a substantially higher yield of structurally informative fragment ions following low-energy CAD of the truncated peptide ion that lacks the arginine residue. In contrast, high-energy CAD yielded superior results for protonated substance P. lo Thus, previous studies suggest that structural features which predispose to localization of charge at a strongly favored site are not conducive to the lowenergy fragmentation of peptide ions via a multiplicity of pathways. The consequence for the effective tandem mass spectrometric characterization of peptides using low-energy CAD is that sequence information may be incomplete. If this argument is correct, then the following specific hypotheses may be proposed: (i) formation of pre-charged derivatives will be detrimental to effective peptide analysis by low-energy CAD. The fixed site of charge will promote only proximal fragmentations, and structural detail remote from the site of charge will not be apparent from the product-ion spectrum. The preparation of pre-charged derivatives of peptides has been advocated as a means of improving ion yield following fast-atom bombardment' and of favoring the detection of particular fragments (retaining the N- or C-terminus, depending on the site of derivatization) following high-energy CAD.^.^. I4-l7 For example, Biemann and coworkers have shown that d-series ions (corresponding to concomitant peptide bond and sidechain cleavages, with charge retention on the N-terminal fragment) are favored during high-energy CAD of peptide ions incorporating a pre-charged N-terminal derivative group.I6 Formation of these ions Received 4 September 1992 Accepted 4 September I992
CHARGE PROMOTION OF LOW ENERGY FRAGMENTATIONS
necessarily occurs remote to the site of charge; it is generally considered that such charge-remote fragmentations have a relatively high energy requiremenP and are therefore not expected as products of low-energy decompositions. Low-energy CAD product-ion spectra of pre-charged derivatives of peptides are therefore predicted to reflect poor yields of sequence-specific fragment ions. (ii) Incorporation of a second charge into ions in which the first charge is fixed (either by a strongly favored site for protonation or by a precharged derivative group) will promote additional diagnostic fragmentation by providing additional possibilities of charge-proximal cleavages. The present report describes analyses designed to assess these specific hypotheses.
659
n
on RI
R,
I ch=c=N-o I'
Pre-charged N-terminal derivative
Pre-charged C-terminal derivative
Scheme 1
EXPERIMENTAL Materials. All peptides are designated using the standard single letter codes for the amino acid residues. GFLCGHYR was provided by D r R. Cook (Baylor College of Medicine, Houston, TX, USA). SIGSLAK and FVQWLMNT (glucagon, 22-29) were purchased from Sigma (St Louis, MO, USA) and Bachem (Torrance, CA, USA), respectively. N- Terminal derivatization of peptides. The procedure adopted was that described by Wagner et aI.l4 Bromoethyl-triphenylphosphonium bromide was prepared by addition of triphenylphosphine (220 mg) to dibromoethane (1 mL). The mixture was stirred at 80 "C for 22 h. The precipitate was washed three times with diethyl ether, dried and stored under nitrogen. The lyophilized peptide (100-300 nmol) was dissolved in 0.16 M potassium borate buffer (pH 9.0; 1mL). To the peptide solution was added a 20-fold molar excess of bromoethyl-triphenylphosphonium bromide dissolved in acetonitrile. The solution was vortex-mixed and allowed to react at 37 "C for 3 h. The reaction mixture was loaded on a Sep-Pak octadecylsilyl silica cartridge (Waters Division of Millipore, Milford, MA, USA). The cartridge was eluted with water (5 mL) and acetonitrile water ( l / l ; 10 mL); the latter fraction, containing the peptide derivative, was dried for subsequent mass spectrometric analysis. C-Terminal deriuatization of peptides. The procedure of Wagner et aI.l4 was used. Aminoethyl-triphenylphosphonium bromide was prepared by solution of bromotriphenylphosphonium bromide (50 mg) in absolute ethanol (250pL) and dropwise addition of ammonium hydroxide (70 pL). The mixture was stirred for 1 h at 20°C and evaporated under a stream of nitrogen. The lyophilized peptide (100-300 nmol) was dissolved in a dilute aqueous solution of trifluoroacetic acid (pH 5.0) and a 20-fold molar excess of dicyclohexylcarbodiimide was added to the solution. The solution was vortex-mixed and heated at 37 "C for 5 min. A 20fold molar excess (over the peptide) of aminoethyltriphenylphosphonium bromide was added to the solution and reaction was allowed to proceed for 3 h at 37 "C. The reaction mixture was lyophilized. Mass spectrometry. All analyses were peformed using a ZAB SEQ hybrid mass spectrometer (VG Analytical, Manchester, UK) with the configuration, BEqQ (where B = magnetic sector, E = electric sector, q = RF-only quadrupole collison region, Q =
+
quadrupole mass filter). Ionization by fast-atom bombardment (FAB) used xenon atoms with energies of 8 k e V as the primary beam. The matrix was a 1 / 1 mixture of thioglycerol and 2,2'-dithiodiethanol, saturated with oxalic acid. Low-energy collisional activation (in q) used argon as collision gas at an estimated pressure (in the collision region) of 1.8 - 3.4 X mBar, achieving 50-60% attenuation of the precursor ion beam. The collision energy was 10-15 eV. Product-ion spectra were acquired via the VG 11-250 data system in the 'multichannel analyzer' mode. The nomenclature used for the peptide fragment ions is that suggested by Roepstorff and F ~ h l m a n , extended '~ (as described in the Results and Discussion section) to the description of the products of pre-charged derivatives. RESULTS AND DISCUSSION The role of the charge site in determining the extent and pathways of low-energy decompositions of peptides has been examined by comparison of the production spectra of protonated peptides and of pre-charged derivatives. We have used the derivatization procedures described by Wagner et al. 14. 's to permit the introduction of a charged functional group (the ethyltriphenylphosphonium moiety) at either the C- or the N-terminus (Scheme 1). Figure 1 shows a comparison of the low-energy CAD product-ion spectra of protonated FVQWLMNT (glucagon 22-29) and the N-terminal ethyl-triphenylphosphonium derivative. The protonated peptide yields prominent B-series sequence ions, accompanied by Y-series ions of lower abundance. The favorable low-energy fragmentation properties of this protonated peptide are interpreted to reflect the lack of a strongly favored site of protonation, with a consequently heterogeneous (with respect to site of charge) precursor-ion population. The product-ion spectrum of the pre-charged derivative (Fig. l(b)) is dominated by the CH2=CH-P+(C6H5)3 fragment; peptide sequence ions ('A- and 'B-series) are observed with very low signal-to-noise ratios. (Although the nomenclature used for the peptide fragments is based on the recommendations of Roepstorff and Fohlman, for the pre-charged derivatives, hydrogen additions or deletions from the fragment ions are shown relative to the pre-charged precursor; the equivalent nomenclature for the protonated . peptides is conventionally shown relative to the neutral molecule.) Thus, prep-
660
CHARGE PROMOTION O F LOW ENERGY FRAGMENTATIONS 1038
M+ 1326
Figure 1. Product-ion spectra recorded following low-energy CAD of precursors derived from the octapeptide, FVQWLMNT. (a) The protonated peptide, [M + HI', mlz 1038. (b) The pre-charged N-terminal ethyl-triphenylphosphonium derivative, M', m / z 1326.
aration of a pre-charged derivative is detrimental to the abundances of structurally diagnostic fragment ions. Figure 2(a) and (b) shows the spectra obtained by low-energy CAD of the protonated heptapeptide, SIGSLAK, and of the C-terminal ethyl-triphenylphosphonium derivative of the corresponding amide, respectively. The spectrum of the protonated peptide contains extensive sequence information; all members of the Y-series are present and B2-B, ions are also observed. In addition, prominent ions corresponding to loss of water from B-series ions are present. The diverse fragmentations of this protonated peptide are again attributed to the lack of a strongly favored site of protonation, resulting in a heterogeneous (with respect to site of charge) precursor-ion population. The slightly greater prominence of fragment ions retaining the C-terminus (though this is not pronounced) is taken to result from the stabilization of product ions which include the somewhat basic lysine residue. The product-ion spectrum (Fig. 2( b)) of the pre-charged derivative (now located at the C-terminus) is again dominated by a fragment, CH2=CH-P+(C6H5)3, associated with the derivative group. No sequence-related product ion exceeds 2.5% of the relative abundance of this fragment; Yi - Yi ions are, however, detectable. The yields of sequence-specific product ions from SIGSLAK [M + H]+ and from the SIGSLAK precharged derivative M + ions were compared by recording spectra obtained with similar precursor-ion abundances. This comparison indicated, for example, that the Y; ions derived from the native peptide were detected with a signal-to-noise ratio approximately six times that observed for the Y; ion derived from the pre-charged derivative. The observation (albeit at very low abundance) of the products of apparent charge-remote processes in the spectra of the pre-charged derivatives (Y'-series
ions in the case of thue C-terminal derivative, and principally 'A-series ions for the example of the N-terminal derivative) may have two explanations. (i) The peptide sequence ions may indeed result from
IM+W Y5" 475
675
I
Figure 2. Product-ion spectra recorded following low-energy CAD of precursors derived from the heptapeptide, SIGSLAK. (a) The protonated peptide, [M+H]+, mlz 675. (b) The pre-charged C-terminal ethyl-triphenylphosphonium derivative of the amide analogue, M+, m / z 962. (c) The protonated, pre-charged derivative, [M+H]'+, mlz4$1.5. m l z ratios are rounded down to the nearest integral or half-integral value.
CHARGE PROMOTlON OF LOW ENERGY FRAGMENTATIONS
66 1
Figure 3. Product-ion spectra recorded following low-energy CAD of precursor derived from the octapeptide, GFLCGHYR. (a) The protonated peptide, [M+ HI+, mlz 952. (b) The doubly protonated peptide, [M+2H12+, mlz476.5. mlz Ratios are rounded down to the nearest integral or half-integral value. The assignment of mlz 318 as the doubly charged ion, Y'y, rather than thc singly charged B, (which would have a similar m / z ratio), is preferred on the basis of the peak width apparent in the expanded spectrum.
charge-remote fragmentation of a very minor subpopulation of precursor ions of sufficiently high internal energy. Wagner et ~ 1 . 'l5 ~ have . previously noted the presence of 'A ions in the high-energy C A D production spectra of N-terminal ethyl-triphenylphosphonium derivatives of peptides; the equivalent C-terminal derivatives, however, yielded 'Y product ions following high-energy CAD. Thus, there are mechanistic differences, yet to be elucidated, between the two decomposition regimes. (ii) The peptide sequence ions may be associated with charge proximal processes resulting from the adoption of the appropriate conformation of the gas-phase precursor ion. (Previous studies have highlighted the significance of gas-phase conformation in determining low-energy fragmentations of peptides.'*. 2") The most striking feature of the spectra of the precharged derivatives, however, is the very low relative abundances of sequence-specific, low-energy fragmentations, consistent with the predicted detrimental effect of the fixed site of charge. These data are therefore in accord with our initial hypothesis that multiple lowenergy decompositions of peptides are promoted by a heterogeneous (with respect to site of charge) population of precursor ions, with the corollary that a fixed site of charge affords unfavorable fragmentation properties. Extending this same logic suggests that protonation of a pre-charged peptide derivative (yielding a singly protonated, but doubly charged, ion) would restore the propensity to fragment via multiple pathways. Figure 2(c) shows the product-ion spectrum of the protonated, pre-charged derivative of SIGSLAK. The C H d H - P + ( C , H , ) , fragment ion associated with the pre-charged derivative group now appears with low relative abundance (in contrast to the spec-
trum of the singly charged derivative, Fig. 2(b)) and peptide sequence ions (both C- and N-terminal) assume much greater prominence. In particular, fragment ions resulting from cleavage between the isoleucine and glycine residues (yielding Y;, Y!;, and B2 (and the associated A2 product ion)) are prominent, suggesting that Coulombic repulsion is effective in favoring a site of protonation which promotes these cleavages via charge proximal mechanisms. The failure to observe B , , YA or Yl fragment ions, however, which would be predicted to result from protonation of the serine/ isoleucine peptide bond, suggests mechanistic complexities which require further investigation. In addition, the absence of an ion of m / z 674, complementary to the derivative-related ion of m / z 289, may indicate a significant contribution from sequential fragmentation processes*' which might be probed by tandem mass spectrometric (MS/MS/MS) techniques. The same principles apply to a comparison of the product-ion spectra obtained by low-energy C A D of singly and doubly protonated GFLCGHYR (Fig. 3). The spectrum of the singly protonated cysteinecontaining analogue is shown in Fig. 3(a). The spectrum is dominated by the Yy ion, reflecting the heavily favored charge location at the strongly basic arginine residue (though the presence of other sequence ions of low relative abundance may suggest minor proportions of the precursor population which incorporate protonation at alternative sites). As previously reported, superior fragmentation properties were observed for the protonated analogue in which the cysteine residue is oxidized to cysteic acid; the effect was attributed to a reduced propensity for charge localization on the arginine residue resulting from interaction with the cysteic acid sidechain. The FAB mass spectrum of the
662
CHARGE PROMOTION O F LOW ENERGY FRAGMENTATIONS
cysteine-containing peptide (GFLCGHYR) includes a weak signal corresponding to the doubly protonated molecule. Despite its low abundance, selection of the doubly protonated precursor during tandem mass spectrometric analysis is facilitated by its appearance at approximately half-integral mass. The product-ion spectrum (Fig. 3(b)) shows prominent cleavages occurring remote from the arginine residue, consistent with the notion that charge repulsion favors a second site of protonation distant from the presumed most favored site (the arginine residue). Thus, cleavages of the phenylalanine/leucine bond (yielding B2 and Y;, Y 0’) and the leucine/cysteine bond (yielding Yy, YI;’) are strongly promoted. (The possible fragment ions, B3and Y r;r, both arising from cleavage of the leucine/cysteine bond, have similar mlz ratios. The assignment of rnlz 318 to Y’; (that is, a doubly charged ion) is tentatively preferred based on the peak width apparent from the expanded spectrum (not shown). The relative contributions of B, and Yy’fragments to the signal at mlz 318 do not, however, affect the validity of the argument that cleavage of the leucine/cysteine bond is promoted by proximal protonation.) Interestingly, fragmentation of the peptide bond between residues 1 and 2 (glycine and phenylalanine) is not observed for the doubly protonated peptide (Fig. 3(b)), an observation that parallels that made for the protoanted, pre-charged derivative of SIGSLAK (Fig. 2(c)). The tandem mass spectrometric analysis of peptides containing a C-terminal arginine residue is a common requirement in protein characterization because of the frequent observation of such peptides in mixtures derived from protein hydrolyses using the enzyme, trypsin. Electrospray-tandem mass spectrometry, involving the low-energy C A D of doubly protonated precursors, has been particularly effective for the analysis of tryptic digest^.'^-^' This success may be attributed, in part, to the promotion of peptide ion fragmentation by the second proton, the first being strongly associated with the arginine residue. CONCLUSION The evidence of the unfavorable low-energy fragmentation properties of pre-charged derivatives of peptides reinforces the view that the promotion of multiple diagnostic fragmentations by low-energy CAD of peptide ions is contingent upon a precursor-ion population that is heterogeneous with respect to the location of charge. Equivalently, native or introduced structural features which lead to a single preponderant site of charge are detrimental to the generation of informative product-ion spectra under low-energy CAD conditions. When protonation of a peptide takes place at a single
strongly favored site (such as the C-terminal arginine residue frequently observed for peptides derived by tryptic hydrolysis of proteins), more favorable fragmentation properties may be observed for the doubly protonated analogue.
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