Improving quantitative glycoproteomics by systematic evaluation of different glycopeptide enrichment, quantification, and mass spectrometry strategies

In this study, we conducted a systematic investigation into a glycoproteomic workflow. Firstly, Label-free and TMT labeling strategies were utilized for intact N-glycopeptide quantification. A total of 2,924 unique intact N-glycopeptides can be quantified after being labeled using TMT approach. Compared to label-free method, TMT labeling for its high precision with the CV averaging 8% and multiplex analysis capabilities. Subsequently, we compared the capacity of ZIC-HILIC and MAX enrichment methods, revealing that the ZIC- HILIC method demonstrated superior performance in enriching intact N-glycopeptides, which can identify 3,004 GPSMs from a single injection of patient-derived xenograft (PDX) sample. Furthermore, we systematically evaluated different higher-energy collisional dissociation (HCD) collision energies, determining that the stepped collision energy HCD (sceHCD) settings of 25, 35, and 45 was the optimal for both the identification and quantification of intact N-glycopeptides.