Introduction:
Protein glycosylation is a critical post-translational modification that affects protein structure, stability, and function. However, the high degree of heterogeneity of glycan structures means that the abundance of each glycoform is relatively low. Typically, mass spectrometry (MS) instrument sensitivity is a significant limitation when analyzing glycopeptides. Thus, challenges associated with low glycopeptide abundances can be overcome with strategies like glycopeptide enrichment, a lower flow rate separation regime, and utilization of higher MS/MS sensitivity systems. Furthermore, alternative MS/MS fragmentation techniques, like electron-activated dissociation (EAD), can complement traditional collision-induced dissociation (CID) for extended characterization of glycopeptides.
Methods:
High-abundance proteins were depleted from human plasma using Top14 depletion spin columns. After digestion of the remaining proteins with trypsin, glycopeptides were enriched using BioSPE GlycanClean kits. Nanoflow separation was done with M-Class HPLC system using trap-elute nanoflow (300 nL/min) with a 50min, 100min and 150min reverse-phase gradients on a C18 column. DDA was performed using a ZenoTOF 8600 system, using both CID and EAD fragmentations and MS data were then processed using PEAKS GlycanFinder software.
Results:
The combination of EAD with CID (EAciD) fragmentation and higher sensitivity in MS1 and MS2 delivered by ZenoTOF 8600 system resulted in 1,972 glycopeptides being identified in 10 µg of N-linked glycopeptide enriched and depleted plasma digest This represents a significant increase of >50% over previously reported results. High MS/MS signal intensity enabled improved identification of glycopeptides from less sample loads while nanoflow separation further enabled greater depth. Using PEAKS GlycanFinder software to process EAD MS/MS data, the glycopeptide backbones were confidently sequenced using the resulting c and z’ fragment ions. In addition, tunability of EAD retained the intact glycan modifications to determine the glycan structures and exact glycan PTM sites.
Conclusion:
Ultra-high sensitivity MS/MS with EAciD fragmentation improved identification and characterization of more N-linked glycopeptides in human plasma.