Tandem HILIC-IMAC strategy for simultaneous N-glycoproteomics and phosphoproteomics in aging mouse brain

Abnormal glycosylation and phosphorylation are strongly associated with brain aging. N-glycosylation and phosphorylation are closely involved in pathological processes in a crosstalk dependent manner. However, simultaneous characterization of glycosylation and phosphorylation together in aging brain was uncommon. Herein we developed a novel tandem HILIC-IMAC strategy for simultaneous analysis of N-glycoproteomics and phosphoproteomics. This tandem method showed higher enrichment repeatability, more identifications of glycopeptides and phosphopeptides, and lower overlap. Application of the established method to mouse brain at two ages (8 weeks and 65 weeks) to explore changes in glycosylation and phosphorylation during aging. Up to 10,990 N-glycopeptides and 11,409 phosphopeptides were identified from mouse brain. Among these, differentially expressed phosphoproteins were involved in regulation of microtubule depolymerization, synapse, and transmission of nerve impulse. And glycoproteins differentially expressed with age were mostly related to cell adhesion processes and extracellular matrix. Furthermore, we found the opposite expression trends in glycosylation and phosphorylation during aging on Grin2b. Together, the HILIC-IMAC strategy has potential to discover aging biomarkers and analyze complex biosamples, paving the way for in-depth investigations for the changes of protein glycosylation and phosphorylation in aging brain.