Abstract
Mass spectrometry-based unbiased analysis of the full complement of secretory peptides is expected to facilitate the identification of unknown biologically active peptides. However, tandem MS sequencing of endogenous peptides in their native form has proven difficult because they show size heterogeneity and contain multiple internal basic residues, the characteristics not found in peptide fragments 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 collision induced dissociation (CID), to identify endogenous peptides derived from secretory granules of a human endocrine cell line. For mass accuracy, both MS and tandem 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 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. These findings point to the utility of ETD mass spectrometry in the global study of endogenous peptides, or peptidomics.
Highlights
Active peptides, commonly known as peptide hormones and antimicrobial peptides, belong to a defined set
The benefit of electron transfer dissociation (ETD) in bottom-up proteomics has been increasingly documented, whereas endogenous peptides remain largely unexplored by ETD, despite the expectation that ETD would improve sequencing for larger peptides
We describe the merits of using ETD, in connection with collision induced dissociation (CID), in peptidomics studies
Summary
Sample Preparation—The human islet cell line QGP-1 (ϳ1 ϫ 107 cells) (15) was stimulated with 10 M carbachol plus 50 mM potassium chloride in serum-free Hank’s balanced solution. For ETD, the two most intense precursor ions were subjected to MS/MS with an ETD activation time of 90 ms for doubly charged ions. An aliquot of the sample was reductive alkylated using dithiothreitol and iodoacetamide to identify peptides derived from chromogranin B (CgB), whose N-terminal region contains two cysteine residues forming a disulfide bond. Antibody Preparation and Mass Spectrometric Characterization of Immunoreactivity—Cysteinyl C-terminal 13-residue peptide of human VGF[554 –577]-NH2 (CHYHHALPPSRHYP-NH2) was conjugated with maleimide-activated keyhole limpet hemocyanin (Thermo Fisher Scientific). 200 l of 3% tryptosoy broth containing 10% alamarBlueTM was added to the reaction mixture and further incubated for the period of time shown in parentheses: E. hirae, S. aureus 209P and E. coli B (4 h), E. coli K12 and E. coli kp (6 h), M. luteus (7 h), S. saprophyticus KD (7.5 h), and P. pastoris GS115 (20 h). Cathelicidin and -defensin-2 (Peptide Institute, Osaka, Japan) were used as control
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