Abstract
The carboxyl groups of tryptic peptides were derivatized with a tertiary or quaternary amine labeling reagent to generate more highly charged peptide ions that fragment efficiently by electron transfer dissociation (ETD). All peptide carboxyl groups—aspartic and glutamic acid side-chains as well as C-termini—were derivatized with an average reaction efficiency of 99 %. This nearly complete labeling avoids making complex peptide mixtures even more complex because of partially-labeled products, and it allows the use of static modifications during database searching. Alkyl tertiary amines were found to be the optimal labeling reagent among the four types tested. Charge states are substantially higher for derivatized peptides: a modified tryptic digest of bovine serum albumin (BSA) generates ~90% of its precursor ions with z > 2, compared with less than 40 % for the unmodified sample. The increased charge density of modified peptide ions yields highly efficient ETD fragmentation, leading to many additional peptide identifications and higher sequence coverage (e.g., 70 % for modified versus only 43 % for unmodified BSA). The utility of this labeling strategy was demonstrated on a tryptic digest of ribosomal proteins isolated from yeast cells. Peptide derivatization of this sample produced an increase in the number of identified proteins, a >50 % increase in the sequence coverage of these proteins, and a doubling of the number of peptide spectral matches. This carboxyl derivatization strategy greatly improves proteome coverage obtained from ETD-MS/MS of tryptic digests, and we anticipate that it will also enhance identification and localization of post-translational modifications.
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Acknowledgments
The authors thank Amelia (Mia) Zutz for performing labeling reactions on the neurotensin peptide standards, M. Violet Lee for compiling the precursor charge state data, and A. J. Bureta for help with figure illustrations. The authors are grateful to Professor Toshifumi Inada at Nagoya University, Nagoya, Japan for the gift of the YIT613 FLAG-tagged yeast strain. This work was supported by the National Institutes of Health: NIGMS Program Project P01GM081629, R01 GM080148, and NHGRI Center of Excellence in Genomic Science 1P50HG004952.
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Frey, B.L., Ladror, D.T., Sondalle, S.B. et al. Chemical Derivatization of Peptide Carboxyl Groups for Highly Efficient Electron Transfer Dissociation. J. Am. Soc. Mass Spectrom. 24, 1710–1721 (2013). https://doi.org/10.1007/s13361-013-0701-2
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DOI: https://doi.org/10.1007/s13361-013-0701-2