Methods to improve quantitative glycoprotein coverage from bottom‐up LC‐MS data

Abstract Advances in mass spectrometry instrumentation, methods development, and bioinformatics have greatly improved the ease and accuracy of site‐specific, quantitative glycoproteomics analysis. Data‐dependent acquisition is the most popular method for identification and quantification of glycopeptides; however, complete coverage of glycosylation site glycoforms remains elusive with this method. Targeted acquisition methods improve the precision and accuracy of quantification, but at the cost of throughput and discoverability. Data‐independent acquisition (DIA) holds great promise for more complete and highly quantitative site‐specific glycoproteomics analysis, while maintaining the ability to discover novel glycopeptides without prior knowledge. We review additional features that can be used to increase selectivity and coverage to the DIA workflow: retention time modeling, which would simplify the interpretation of complex tandem mass spectra, and ion mobility separation, which would maximize the sampling of all precursors at a giving chromatographic retention time. The instrumentation and bioinformatics to incorporate these features into glycoproteomics analysis exist. These improvements in quantitative, site‐specific analysis will enable researchers to assess glycosylation similarity in related biological systems, answering new questions about the interplay between glycosylation state and biological function.