In-depth plasma N-glycoproteome profiling using narrow-window data-independent acquisition on the Orbitrap Astral mass spectrometer - Communications
Summary
To set up a glycoproteomics-directed narrow-window DIA method, i.e., nGlycoDIA, several experimental parameters needed to be considered and optimized, including precursor m/z range, isolation window size, maximum ion injection time, and collision energy. Additionally, as shown for standard proteomics with the introduction of 40 up to 180 samples per day (SPD) methods31, we hypothesized that the increased speed of the Orbitrap Astral MS would allow us to shorten the LC-MS runs, without a significant loss in identifications. This illustrates that the data lost by shifting the precursor m/z range is minimal, and that this rather results in a beneficial additional degree of enrichment in the gas phase, as was demonstrated in the aforementioned comparison with DDA. Taking a closer look at the structure of the hetero-dimer and the glycans, we noticed that the core GlcNAcs from IL-12A-N217 formed hydrogen bonds with the sidechain of the Gln42 residue of IL-12A, which may aid in stabilizing its conformation upon hetero-dimerization (Fig. While the available version of FragPipe was not able to analyze the data in the same way as Byonic, i.e., by identifying the MS1 precursor mass and treating the DIA-data as DDA-data, we were able to make use of two alternative strategies: 1) using DIA-Umpire and 2) using an experimental library-based DIA search.