TIMAHAC: Streamlined Tandem IMAC-HILIC Workflow for Simultaneous and High-Throughput Plant Phosphoproteomics and N-glycoproteomics.
Mol Cell Proteomics
; 23(5): 100762, 2024 May.
Article
in En
| MEDLINE
| ID: mdl-38608839
ABSTRACT
Protein post-translational modifications (PTMs) are crucial in plant cellular processes, particularly in protein folding and signal transduction. N-glycosylation and phosphorylation are notably significant PTMs, playing essential roles in regulating plant responses to environmental stimuli. However, current sequential enrichment methods for simultaneous analysis of phosphoproteome and N-glycoproteome are labor-intensive and time-consuming, limiting their throughput. Addressing this challenge, this study introduces a novel tandem S-Trap-IMAC-HILIC (S-Trap suspension trapping; IMAC immobilized metal ion affinity chromatography; HILIC hydrophilic interaction chromatography) strategy, termed TIMAHAC, for simultaneous analysis of plant phosphoproteomics and N-glycoproteomics. This approach integrates IMAC and HILIC into a tandem tip format, streamlining the enrichment process of phosphopeptides and N-glycopeptides. The key innovation lies in the use of a unified buffer system and an optimized enrichment sequence to enhance efficiency and reproducibility. The applicability of TIMAHAC was demonstrated by analyzing the Arabidopsis phosphoproteome and N-glycoproteome in response to abscisic acid (ABA) treatment. Up to 1954 N-glycopeptides and 11,255 phosphopeptides were identified from Arabidopsis, indicating its scalability for plant tissues. Notably, distinct perturbation patterns were observed in the phosphoproteome and N-glycoproteome, suggesting their unique contributions to ABA response. Our results reveal that TIMAHAC offers a comprehensive approach to studying complex regulatory mechanisms and PTM interplay in plant biology, paving the way for in-depth investigations into plant signaling networks.
Key words
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Phosphoproteins
/
Chromatography, Affinity
/
Arabidopsis
/
Proteomics
/
Workflow
Language:
En
Journal:
Mol Cell Proteomics
Journal subject:
BIOLOGIA MOLECULAR
/
BIOQUIMICA
Year:
2024
Document type:
Article
Affiliation country:
Taiwán