This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The structure of peptide b-ions has been the subject of a long-standing debate. Early experimental work based on its dissociation behavior suggested the oxazolone structure as the most stable form, a conclusion that is supported by both the theoretical modeling and the IR spectroscopic study. However, there have been growing evidences lately, showing that b-ions may also assume a macro-cyclic form, with its N-terminal amino group forming an amide bond with its C-terminus. The macro ring may reopen, sometimes at a different position from the original one, leading to extensive sequence scramblings and subsequent formation of indirect sequence ions under collision-induced dissociation (CID) conditions. This is problematic for peptide sequencing and protein identification, particularly when multiple stages of CID are involved. The utility of an CID/ECD MS3 approach in protein sequencing and PTM analysis has recently been demonstrated in our laboratory (Theberge 2010, Li 2010). It is thus of practical importance to investigate whether sequence scrambling also presents a problem when ECD is employed instead in the second stage of an MS3 experiment. In this study, we investigated the ECD behavior of doubly charged b-ions from several peptides with multiple basic residues near their N-termini. ECD of the b8 and b9 ions from alpha-neurokinin (HKTDSFVGLM-NH2) generated a near complete series of c-ions, which seems to be consistent with the oxazolone structure. However, these c-ions comprise only a small portion of the product ions. Most fragment ions can only be explained by sequence scrambling resulting from the formation of a macro ring connecting the C-terminal carbonyl group to the N-terminal amino group, followed by ring openings at different positions. Further, ECD of smaller b-ions from alpha-neurokinin produced predominantly sequence scrambled fragment ions, with no c-ions, indicative of the size influence on b-ion structures. Sequence scramblings were not observed in ECD of any b-ion from eledoisin (pEPSKDAFIGLM-NH2) studied here. The N-terminal amino group of eledoisin is blocked due to the presence of the pyroglutamic acid residue, thus preventing the formation of the cyclic structure. Despite the presence of an unblocked N-terminal amino group, there was little evidence for sequence scrabmlings in ECD of a series of b-ions (b5-b10) from substance P (RPKPQQFFGLM-NH2). However, in addition to the normal c-ion series, ECD of these b-ions also produced a series of fragment ions that correspond to c-ions with additional lysine sidechain loss (c-Lys), which suggested another type of cyclic b-ion structure, where the C-terminal carbonyl group formed an amide bond with the lysine sidechain amino group. The relative abundance of these c-Lys ions in ECD spectra of b ions increases as its size increases from b5 to b7, then decreases from b8 to b10. To better understand the ECD behavior of these b-ions, ab initio-calculations were preformed on two doubly charged b ions (b6 and b8) from substance P to examine the relative stability of oxazolone and cyclic structures. Due to their relatively large sizes, RHF/3-21G level of theory was applied without ZVPE correction. The results showed that for b6 ion, the oxazolone structure and the lysine-linked cyclic structure have similar stability, which explains why both c and c-Lys ions were observed with comparable abundances. On the other hand, the Lys-cyclic structure for b8 ion was predicted to be 15 kcal/mol more stable than the oxazolone form, consistent with the predominance of c-Lys ions over normal c ions in b8 ion ECD. In both cases, the N-terminus cyclic structure were found to be significantly higher in energy than the corresponding Lys-cyclic form (52 kcal/mol for b8 and 28 kcal/mol for b6), which is probably why sequence scrambling was hardly observed here. Finally, ECD on larger b(n) ions (n>20) from a number of proteins were also performed. The preliminary results showed little evidence of sequence scrambling. This might be due to a disfavored entropic factor to bring the N- and C-termini close enough to form the macro ring structure. These results suggested that a peptide b-ion may exist as a mixture of several different forms, with their propensities influenced by its size, N-terminus, and possibly the sidechains of basic amino acid residues. Although indirect sequence ions may also be formed in CID/ECD experiments, this is not expected to be a significant problem for larger b ions. This research will be presented as a poster at the 58th annual ASMS conference (Lin et al. 2010). Update for 2010-2011: A manuscript describing this work, entitled """"""""Structural Heterogeneity of Doubly-Charged Peptide b-Ions"""""""", has been published on Journal of the American Society for Mass Spectrometry, 2011, 22, 245-254.

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