In Brief As a highly abundant and diverse post-translational modification, protein glycosylation is challenging to characterize in various approaches including MS. In MS-based proteomics, data-independent acquisition (DIA) has been advanced rapidly and showed outstanding analytical performances. DIA now started to be applied in different facets of glycoproteomics, including deglycosylated and intact N-linked and O-linked glycopeptides, and screening of oxonium ions. We summarized current applications of DIA in glycoproteomics and discussed its limitations and perspectives.
