Abstract
Phosphorylation and glycosylation are two of the most important and widespread post-translational modifications (PTMs) in an organism. Proteomics analysis of the PTMs has been challenged by low stoichiometry of the modified proteins and suppression effects by high abundance proteins, typically no-functional house-keeping proteins. In this study, a novel method was applied for not only isolating PTM peptides from intact peptides but also concurrently characterizing of glyco- and phospho- proteome using electrostatic repulsion hydrophilic interaction chromatography (ERLIC) packed with silica coated by cross- linked polyethyleneimine. For 2 mg tryptic digest of mouse proteome of epicardial adipose tissue with fat diet, 802 N-glycosy- lated peptides of 316 glycoproteins and 159 phosphorylated peptides of 75 phosphoproteins were identified using HPLC chip/ quadrupole time-of-flight (Q-TOF) tandem mass spectrometer.
Highlights
It is estimated that about 50% of all mammalian proteins are carrying glycan residues,[1] and 30% of all proteins are phosphorylated.[2]
electrostatic repulsion hydrophilic interaction chromatography (ERLIC) Chromatogram According to the principle of peptide fractionation by ERLIC, unmodified peptides hypothetically eluted in early fractions whereas glycopeptides retained in column and come out later
A 2 mg protein extracted from epicardial adipose tissue of 16-week high fat diet mouse was digested and peptides were injected into ERLIC
Summary
It is estimated that about 50% of all mammalian proteins are carrying glycan residues,[1] and 30% of all proteins are phosphorylated.[2]. A novel method was applied for isolating PTM peptides from intact peptides and concurrently characterizing of glyco- and phosphoproteome using electrostatic repulsion hydrophilic interaction chromatography (ERLIC) packed with silica coated by crosslinked polyethyleneimine. For 2 mg tryptic digest of mouse proteome of epicardial adipose tissue with fat diet, 802 N-glycosylated peptides of 316 glycoproteins and 159 phosphorylated peptides of 75 phosphoproteins were identified using HPLC chip/ quadrupole time-of-flight (Q-TOF) tandem mass spectrometer.
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