High-Throughput Simultaneous Quantification of Glycopeptides and Phosphopeptides Enabled by 12-plex DiLeu Isobaric Tags and Dual-Functional Titanium(IV)-IMAC Material

ABSTRACT: Protein glycosylation and phosphorylation are two of the most common post-translational modifications (PTMs). Though they have been indicated to play critical roles in various physiological and pathological processes, effective analytical tools are challenged by low PTM abundance and poor ionization efficiency. Recently, an epoxy-ATP-Ti4+-IMAC material has been developed to enable simultaneous enrichment and separation of common N-glycopeptides, phosphopeptides, and mannose-6-phosphate (M6P) glycopeptides from complex biological samples. To further improve the throughput of our previous method, in this study, we developed an efficient strategy using our custom-developed N,N-dimethyl leucine (DiLeu) isobaric tags to achieve a multiplexed quantitative simultaneous analysis of N-glycosylation and phosphorylation from up to 12 samples for the first time. We further applied this method to investigate N-glycosylation and phosphorylation alterations in APP/PS1 model mouse brain samples and wild-type (WT) controls. With the integration of a one-tube sample processing workflow, 1975 glycopeptides and 1181 phosphopeptides were quantitatively identified from mouse brain. We also unveiled important biological functions regulated by N-glycosylation and phosphorylation, including synapse organization, synaptic membrane and cell adhesion, highlighting the involvement of N-glycosylation and phosphorylation aberrations in APP/PS1 models. Our results provide evidence that the established DiLeu isobaric labeling-epoxy-Ti4+-IMAC method can be used for high-throughput simultaneous quantification of glycosylation and phosphorylation, and their potential crosstalk, showing significant promise for identifying novel therapeutic targets or biomarkers in biological systems.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human) Mus Musculus (mouse)

TISSUE(S): Brain, Cell Culture, Pancreatic Duct

DISEASE(S): Alzheimer's Disease

SUBMITTER: Feixuan Wu  

LAB HEAD: Lingjun Li

PROVIDER: PXD061371 | Pride | 2026-06-29

REPOSITORIES: Pride