TIHAMAC: Streamlined Tandem IMAC-HILIC Workflow for Simultaneous and High-Throughput Plant Phosphoproteomics and N-glycoproteomics Analysis

ABSTRACT: Protein post-translational modifications (PTMs) are pivotal molecular events in plant cells, encompassing processes such as protein folding and enzyme activation. Among these PTMs, N-glycosylation and phosphorylation hold particular importance, as they play essential roles in regulating plant growth and stress responses. The intricate PTM landscape is exemplified by the identification of multiple plant membrane-localized proteins subject to both phosphorylation and N-glycosylation, hinting at potential interplay influencing protein functionality. To investigate the crosstalk between phosphorylation and glycosylation in plant signaling, immobilized metal affinity chromatography (IMAC) and hydrophilic interaction chromatography (HILIC) have been primarily utilized for large-scale phosphopeptide and glycopeptide enrichment, respectively. In this work, we introduce a novel streamlined tandem S-Trap-IMAC-HILIC strategy, termed TIMAHAC, to enable serial enrichment of phosphopeptides and glycopeptides. This approach successfully integrates the two methods through tandem tip enrichment, eliminating the need for buffer exchange and sample transfer. As a result, it significantly minimizes sample loss during serial enrichment steps, and streamlines experimental procedures, ultimately saving time. We demonstrated the efficiency of the TIHAMAC strategy in the glycoproteomics and phosphoproteomics analyses using 200 μg of Arabidopsis digests. Up to 1,954 N-glycopeptides and 11,255 phosphopeptides were identified by timsTOF HT. Remarkably, 69% of N-glycopeptides were consistently quantified with a coefficient of variation less than 20%. Furthermore, more than one-third identified glycoproteins exhibited phosphorylation modifications. These results suggest that the applicability of the TIMAHAC method for studying the potential crosstalk of N-glycoproteome and phosphoproteome within complex plant signaling networks.

INSTRUMENT(S): autoflex

ORGANISM(S): Arabidopsis Thaliana (mouse-ear Cress)

SUBMITTER: Chuan-Chih Hsu  

LAB HEAD: Chuan-Chih Hsu

PROVIDER: PXD047065 | Pride | 2024-04-03

REPOSITORIES: Pride