High-throughput simultaneous quantification of glycopeptides and phosphopeptides enabled by 12-plex DiLeu isobaric...

Protein glycosylation and phosphorylation are critical post-translational modifications (PTMs) that regulate diverse physiological and pathological processes, yet their comprehensive characterization remains challenging due to low abundance and poor ionization efficiency. Recent advances using epoxy-ATP-Ti⁴⁺-IMAC materials have enabled simultaneous enrichment of N-glycopeptides, phosphopeptides, and mannose-6-phosphate glycopeptides. However, high-throughput, multiplexed quantification of these PTMs is still lacking. This work addresses the need for an efficient strategy capable of simultaneously enriching, identifying, and quantitatively comparing glycosylation and phosphorylation across multiple biological samples.

We developed a high-throughput workflow integrating epoxy-Ti⁴⁺-IMAC enrichment with custom N,N-dimethyl leucine (DiLeu) isobaric tags to achieve 12-plex quantitative analysis of N-glycosylation and phosphorylation for the first time. This streamlined one-tube sample preparation protocol enabled robust, simultaneous enrichment and quantification of PTMs from complex mouse brain samples. Application to APP/PS1 transgenic mice versus wild-type controls produced quantitative identification of 1975 N-glycopeptides and 1181 phosphopeptides. Comparative profiling revealed substantial PTM alterations associated with Alzheimer's disease (AD)-related pathology. Differentially modified proteins mapped to key biological pathways, including synapse organization, synaptic membrane regulation, and cell adhesion. The abundance patterns highlighted broad disruptions in PTM-mediated signaling and provided molecular insights into synaptic dysfunction in the APP/PS1 model.

This integrated DiLeu isobaric labeling-epoxy-Ti⁴⁺-IMAC platform provides a powerful, high-throughput solution for the simultaneous quantification of glycosylation and phosphorylation, enabling detailed investigation of PTM interplay. By uncovering disease-associated modifications in AD mouse models, this method offers new opportunities to identify mechanistic biomarkers and therapeutic targets. Its versatility and scalability make it broadly applicable to PTM-centric studies across diverse biological systems, including biofluids, cell lysates and tissues.