Large-scale Identification of Endogenous Secretory Peptides Using Electron Transfer Dissociation Mass Spectrometry

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]-NH 2 , 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.

Human tear proteomics and peptidomics in ophthalmology: Toward the translation of proteomic biomarkers into clinical practice

“…Importantly, these naturally cleaved peptides can be bioactive and fulfill specific functions not ascribed to their original protein parents. Such molecules might play important roles in many, if not all, biological processes (such as antimicrobial activity or intercellular signaling) (40)(41)(42)(43)(44)(45). The naturally cleaved peptides have attracted a lot of interest as potential target molecules.…”

Simultaneous quantification of intracellular and secreted active and inactive glucagon-like peptide-1 from cultured cells

“…The flow rates for the first and second dimensions were 20 ll/min and 200 ll/ min, respectively. The MS conditions used for the secretopeptidome analysis were as described by Sasaki and coworkers [26][27][28]. The mass spectrometer was operated using a data-dependent acquisition (DDA) mode.…”

Peptidomics study of anthocyanin-rich juice of elderberry

“…The four most intense ions were chosen for CID fragmentation. Automatic gain control was used to accumulate sufficient fragment ions (MS/MS target value 2E5; maximum injection time, 1000 ms) [9]. A sample blank was run before the analytical sample.…”

“…The combination of high performance liquid chromatography (HPLC) coupled with MS/MS can help to overcome complex peptide mixtures and aid in the identification of less abundant peptides. The use of nano-liquid chromatography can increase the signal-to-noise ratio and sensitivity of MS for the detection of peptides in complex mixtures or those present in low concentrations [9][10][11][12].…”