Analysis of PNGase F‐Resistant N‐Glycopeptides Using SugarQb for Proteome Discoverer 2.1 Reveals Cryptic Substrate Specificities

SugarQb (http://www.imba.oeaw.ac.at/sugarqb) is a freely available collection of computational tools for the automated identification of intact glycopeptides from high‐resolution HCD MS/MS datasets in the Proteome Discoverer environment. We report the migration of SugarQb to the latest and free version of Proteome Discoverer 2.1, and apply it to the analysis of PNGase F‐resistant N‐glycopeptides from mouse embryonic stem cells. The analysis of intact glycopeptides highlights unexpected technical limitations to PNGase F‐dependent glycoproteomic workflows at the proteome level, and warrants a critical reinterpretation of seminal datasets in the context of N‐glycosylation‐site prediction.