Glycan size and attachment site location affect electron transfer dissociation (ETD) fragmentation and automated glycopeptide identification.

We established a small synthetic N-glycopeptide library to systematically evaluate the effect of glycosylation site location and glycan size on the efficiency of electron transfer dissociation (ETD) fragmentation and subsequent automated identification. The glycopeptides within this library differed in glycosylation site position and glycan size ranging from the pentasaccharide N-glycan core to fully sialylated, biantennary N-glycans. Factors such as glycan size, glycosylation site position within a glycopeptide and individual precursor m/z all significantly impacted the number and quality of assignable glycopeptide backbone fragments. Generally, high charge/low m/z precursors (>3+) and glycopeptides carrying neutral, smaller N-glycans gave better product ion spectra, while hardly any product ions were detectable for sialylated, triply charged N-glycopeptides. These factors impacted correct glycopeptide identification by proteomics software tools such as SEQUEST or Amanda. A better understanding how glycopeptide physico-chemical properties influence fragmentation will help optimizing fragmentation conditions and generate better data, which will facilitate software assisted glycopeptide data analyses.