Analysis of the extreme diversity of salivary alpha-amylase isoforms generated by physiological proteolysis using liquid chromatography–tandem mass spectrometry

Abstract

Saliva is a crucial biofluid for oral health and is also of increasing importance as a noninvasive source of disease biomarkers. Salivary alpha-amylase is an abundant protein in saliva, and changes in amylase expression have been previously associated with a variety of diseases and conditions. Salivary alpha-amylase is subject to a high diversity of posttranslational modifications, including physiological proteolysis in the oral cavity. Here we developed methodology for rapid sample preparation and non-targeted LC-ESI-MS/MS analysis of saliva from healthy subjects and observed an extreme diversity of alpha-amylase proteolytic isoforms. Our results emphasize the importance of consideration of posttranslational events such as proteolysis in proteomic studies, biomarker discovery and validation, particularly in saliva.

Circulating Fragments of N-Terminal Pro–B-Type Natriuretic Peptides in Plasma of Heart Failure Patients

“…These semitryptic peptides were most likely due to physiological cleavage at the N and C termini of circulating NT-proBNP before IP (22 ). The identity of the protease or proteases responsible for these cleavage events in NT-proBNP is not clear, nor is whether the cleavage events occur before or after cleavage of proBNP to NT-proBNP and BNP.…”

Oligosaccharyltransferase Subunits Bind Polypeptide Substrate to Locally Enhance N-glycosylation

“…Mass Spectrometry and Data Analysis-Peptides were analyzed via LC-ESI-MS/MS using a Prominence nanoLC system (Shimadzu, Kyoto, Japan) and TripleTof 5600 mass spectrometer with a Nanospray III interface (AB SCIEX, Washington, D.C.) as described (21,22). Glycosylation occupancy was measured at previously identified glycosylation sites (15, 21) using unmodified and GlcNAc-modified versions of the same peptide.…”

Mixed disulfide formation in vitro between a glycoprotein substrate and yeast oligosaccharyltransferase subunits Ost3p and Ost6p

“…Peptides were analysed by LC-ESI-MS/MS using a Prominence nanoLC system (Shimadzu) and TripleTof 5600 mass spectrometer with a Nanospray III interface (AB SCIEX), as previously described [35,36]. Peptides were identified using ProteinPilot (AB SCIEX), searching the LudwigNR database (downloaded from http://apcf.edu.au as at 27 January 2012; 16,818,973 sequences; 5,891,363,821 residues) with standard settings: Sample type, identification; Cysteine alkylation, acrylamide; Instrument, TripleTof 5600; Species, not limited; ID focus, biological modifications; Enzyme, trypsin; Search effort, thorough ID.…”