Quantitative mapping of glycoprotein micro-heterogeneity and macro-heterogeneity: an evaluation of mass spectrometry signal strengths using synthetic peptides and glycopeptides.

摘要: Mass spectrometry (MS) is used to quantify the relative distribution of glycans attached particular protein glycosylation sites (micro-heterogeneity) and evaluate molar site occupancy (macro-heterogeneity) in glycoproteomics. However, accuracy MS for such quantitative measurements remains be clarified. As a key step towards this goal, panel related tryptic peptides with without complex, biantennary, disialylated N-glycans was chemically synthesised by solid-phase peptide synthesis. Peptides mimicking those resulting from enzymatic deglycosylation using PNGase F/A endo D/F/H were synthetically produced, carrying aspartic acid N-acetylglucosamine-linked asparagine residues, respectively, at site. The ionisation/detection strengths these pure, well-defined quantified compounds investigated various ionisation techniques mass analysers (ESI-IT, ESI-Q-TOF, MALDI-TOF, ESI/MALDI-FT-ICR-MS). Depending on ion source/mass analyser, glycopeptides complex-type exhibited clearly lower signal (10-50% an unglycosylated peptide) when equimolar amounts analysed. Less bias observed analysed nano-ESI medium-pressure MALDI. position within also influenced response, if detected as singly or doubly charged signals. This first study systematically quantitatively address determine glycopeptide glycoprotein micro-heterogeneity macro-heterogeneity label-free approaches. These data form much needed knowledge base accurate