Fig. 2: In-depth characterization of the O-glycoproteome in testes of 24-day-old and 12-week-old mice.

a Schematic workflow for analyzing the testis O-glycoproteome. Proteins from testes of 24-day-old and 12-week-old mice were extracted, digested by trypsin, and subsequently de-sialylated using neuraminidase. The O-glycopeptides with Tn and T structures were enriched using VVA and PNA lectins respectively, and then identified by LC-MS/MS using higher-energy dissociation product ions-triggered electron-transfer/higher-energy collision dissociation (HCD-pd-EThcD) strategy. Four biological replicates were used for each group. Illustrations used elements from Servier Medical Art (http://smart.servier.com/) under a Creative Common Attribution 3.0 Generic License (https://creativecommons.org/licenses/by/3.0/). b The numbers of O-glycoproteins, unique O-glycopeptides, unambiguous O-glycosites, and glycopeptide-spectrum matches (GPSM) identified in each group. c Venn diagram showing the distribution of identified O-glycoproteins, O-glycopeptides, and O-glycosites enriched from testes at different developmental stages using different lectins. d, e Overlap of the testis O-glycoproteins identified in this study with those from the previously reported mouse O-glycoproteins atlas (d), as well as with the reported O-glycoproteins of the other 10 mouse organs published by Yang et al.³⁵. (e, f) Overlap of human counterparts of the 337 mouse O-glycoproteins with the published human O-glycoproteins which were mainly identified by chemical methods, LWAC or EXoO strategy. VVA vicia villosa agglutinin, PNA peanut agglutinin, Tn GalNAc-α1-Ser/Thr, T Galβ1,3GalNAc-α1-Ser/Thr, GPSM glycopeptide-spectrum matches, LWAC lectin weak affinity chromatography, EXoO site-specific extraction of O-linked glycopeptides.