Abstract
This protocol outlines the essential steps of mass spectrometry-based analysis of protein samples that can be used to identify post-translational arginylation. We describe special considerations for sample preparation and digestion, mass spectrometry analysis using high-precision instruments, database searching for the addition of N-terminal arginine, and multiple steps of automated and manual data validation. Data validation is especially important and involves automated data filtering, manual elimination of mass ambiguities, isotopic peak checking and analysis of the ion fragmentation patterns of the putative arginylated peptides. This protocol is an unambiguous method for the identification of post-translationally arginylated proteins, and it can be used to identify new arginylated proteins. This method provides a definitive answer on the identity of arginylated proteins and peptides in simple and complex protein preparations. It is recommended for studies of arginylation and can be applied to other poorly understood post-translational modifications.
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Acknowledgements
We thank X. Han for performing some of the searches during the final stages of the development of the automated filtering criteria for arginylated peptides, D. Cociorva for help with optimizing DTASelect filtering conditions, members of the Kashina lab, S. Saha, J. Wang, R. Rai and F. Zhang for preparing the samples used in the mass spectrometry runs that defined the procedure described above. This study was supported by an NIH grant 1R01HL084419-01A1, U.S. Army Research Program Award W81XWH-04-1-0879, a research award from W.W. Smith Charitable Trust to A.K. and by NIH grants P41 RR11823-09 and 5R01 MH067880 to J.R.Y.
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Xu, T., Wong, C., Kashina, A. et al. Identification of N-terminally arginylated proteins and peptides by mass spectrometry. Nat Protoc 4, 325–332 (2009). https://doi.org/10.1038/nprot.2008.248
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DOI: https://doi.org/10.1038/nprot.2008.248
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