Maximizing glycoproteomics identification depth in complex mixtures on the timsTOF Pro platform

Glycoproteins play important roles in numerous physiological processes and are often implicated in disease. Analysis of site-specific protein glycobiology through glycoproteomics is evolving rapidly in recent years thanks to hardware and software innovations. Introduction of Parallel Accumulation Serial Fragmentation (PASEF) on hybrid trapped ion mobility time-of-flight instruments combined deep proteome sequencing with separation of (near-)isobaric precursor ions or converging isotope envelopes through ion mobility separation. Despite these advantages, the use of PASEF in integrated glycoproteomics workflows to comprehensively capture the glycoproteome has received little attention. To address this gap, we have developed an integrated methodology using the timsTOF Pro2 to enhance N-glycopeptide identifications in complex mixtures. We explored its potential by systematically evaluating the impact of ion optics tuning, collision energies, mobility isolation width, and the use of dopant-enriched nitrogen gas (DEN) on glycopeptide identification rates. This comprehensive approach showed a marked increase in unique glycopeptide identification rates compared to standard proteomics settings while evaluating key parameters on a large set of glycopeptides. With short liquid chromatography gradients of 30 minutes, we increased the number of unique N-glycopeptide identifications in full human plasma glycopeptide samples from around 100 identifications under standard proteomics condition to over 1500 with our optimized glycoproteomics approach, highlighting the need for tailored solutions.