In-depth plasma N-glycoproteome profiling using narrow-window data-independent acquisition on the Orbitrap Astral mass spectrometer.

Recently, a conceptually new mass analyzer was introduced by pairing a quadrupole Orbitrap mass spectrometer with an asymmetric track lossless (Astral™) analyzer. This system provides >200 Hz MS/MS scanning speed, high resolving power, sensitivity, and mass accuracy. Due to its speed, the instrument allows for a narrow-window data-independent acquisition (nDIA) strategy, representing a new technical milestone in peptide-centric proteomics. However, this new system may also be applied to other complex and clinically important proteomes, such as the human plasma N-glycoproteome. Here, we evaluate the Orbitrap Astral mass spectrometer for the in-depth analysis of the plasma N-glycoproteome and pioneer a dedicated nDIA workflow, termed "nGlycoDIA", on glycopeptide enriched and crude plasma. This strategy leads to the cumulative identification of over 3000 unique glycoPSMs derived from 181 glycoproteins in just 40 minutes and covers a dynamic range of 7 orders of magnitude for a glycopeptide enriched plasma sample. Notably, we detect several glycosylated cytokines that have reported plasma concentrations in the ng/L range. Furthermore, shortening the gradient to 10 min still allows for the detection of almost 1850 (95% CI [1840-1860]) unique glycoPSMs, indicating that high-throughput in-depth clinical plasma glycoproteomics may be within reach.