Abstract 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 &gt;200-Hz MS/MS scanning speed, high resolving power and sensitivity, and low-ppm mass accuracy. This instrument allows a narrow-window data-independent (nDIA) strategy, improving sensitivity and reproducibility even when using very short LC gradients. Although this represents a new technical milestone in peptide-centric proteomics, this new system has not yet been evaluated for the analyses of very complex and clinically important proteomes, such as represented by the plasma glycoproteome. Here, we evaluated the Orbitrap Astral mass spectrometer for the analysis of the plasma glycoproteome, and pioneer a dedicated nDIA workflow, themed nGlycoDIA. With substantially adjusted parameters and varying collision energies, nGlycoDIA has clear benefits for plasma glycoproteomics. We tested our method both in glycopeptide enriched and crude plasma, leading to the identification of more than 3000 unique glycoPSMs from 181 glycoproteins, covering a dynamic range of 7 orders of magnitude in the enriched plasma sample in just 40 minutes. In addition, we detect for the first time several glycosylated cytokines that have a reported plasma concentration in the ng/L range. Furthermore, shortening the gradient to 10 min still allows the detection of almost 2500 unique glycoPSMs from enriched plasma, indicating that high-throughput indepth clinical plasma glycoproteomics may be within reach.

Narrow window data-independent acquisition on the Orbitrap Astral Mass Spectrometer enables fast and deep coverage of the plasma glycoproteome