Amino acid substitutions to viral proteins can create or remove glycosites. While research groups have published assignment of viral protein glycosylation, there remains little consensus regarding how to quantify the glycosylation changes that occur among viral variants. This is because glycosylation is inherently micro- and macro-heterogeneous, making rigorous comparison of the complete glycosylated structures of viral proteins a statistical problem. In response, we have compared glycoproteomics data acquisition and bioinformatics methods for producing confident measurements of glycosylation similarity. We compared glycoproteomics assignments and quantification from data acquired with data-dependent acquisition (DDA), stepped window data-independent acquisition (swDIA), and broad mass range data-independent acquisition coupled with ion mobility spectrometry (HDMSE), respectively.<br/><br/>We compared DDA, swDIA, and HDMSE mass spectral data to assign and quantify (i) the five <i>N</i>-linked glycosylation sites of the glycoprotein standard alpha-1-acid glycoprotein (AGP), (ii) the 12 sites of an influenza A virus hemagglutinin (HA) and (iii) the 22 sites of SARS-CoV-2 spike protein. For all three proteins, we observed that swDIA provided greater depth of coverage for glycopeptide precursor ions compared with DDA. The performance improvement of swDIA was mitigated to a degree by the difficulty of assigning low abundance precursor ions confidently. For this reason, we compared the performance of HDMSE data acquired using the Waters Cyclic IMS instrument, for which there is no precursor isolation step and no need for scanned quadrupole windows. The Cyclic IMS instrument alternated scans corresponding to low and high collision energy in a collision cell located after the mobility chamber. The resulting collision energy aligned retention time curves contained no missing data. We developed a glycopeptide-aware deconvolution approach to assign the HDMSE data accurately. For this, we connected precursors and product ions according to the combined retention time (RT) and ion mobility (IM) profiles . Using this approach, we demonstrated that HDMSE improved the coverage of glycopeptides over swDIA and DDA.

Comparison of MS acquisition modes for assigning and quantifying glycopeptides

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ACS Fall 2022

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