Large-Scale Identification of Endogenous Secretory Peptides using Electron Transfer Dissociation Mass Spectrometry
News Jan 09, 2013
Mass spectrometry-based unbiased analysis of the full complement of secretory peptides is expected to facilitate the identification of unknown biologically active peptides. However, MS/MS sequencing of endogenous peptides in their native form has proven difficult since they show size heterogeneity and contain multiple internal basic residues, the characteristics not found in peptides produced by in vitro digestion. Endogenous peptides remain largely unexplored by electron transfer dissociation (ETD), despite its widespread use in bottom-up proteomics. We used ETD, in comparison to CID, to identify endogenous peptides that were derived from secretory granules of a human endocrine cell line. For mass accuracy, both MS and MS/MS were analyzed on an Orbitrap. CID and ETD, performed in different LC-MS runs, resulted in the identification of 795 and 569 unique peptides (ranging from 1000 to 15000 Da), respectively, with an overlap of 397. Peptides larger than 3000 Da accounted for 54% in CID and 46% in ETD identifications. Although numerically outperformed by CID, ETD provided more extensive fragmentation, leading to the identification of peptides that are not reached by CID. This advantage was demonstrated in identifying a new antimicrobial peptide from neurosecretory protein VGF (non-acronymic), VGF[554-577]-NH2, or in differentiating nearly isobaric peptides (mass difference being less than 2 ppm) that arise from alternatively spliced exons of the gastrin-releasing peptide gene. CID and ETD complemented each other to add to our knowledge of the proteolytic processing sites of proteins implicated in the regulated secretory pathway. An advantage of the use of both fragmentation methods was also noted in localization of phosphorylation sites.
The study is published online in the journal Molecular & Cellular Proteomics and is free to access.
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