Cotton Ti-IMAC: Developing Phosphorylated Cotton as a Novel Platform for Phosphopeptide Enrichment

ABSTRACT: Protein phosphorylation is one of the most common post-translational modifications (PTMs), which is involved in many important physiological functions. Understanding protein phosphorylation at molecular level is critical to decipher its relevant biological processes and signaling networks. Mass spectrometry (MS) has been proved to be a powerful tool in comprehensive characterization of protein phosphorylation. Yet the low abundance and poor ionization efficiency of phosphopeptides make its MS analysis challenging; an enrichment with high efficiency and selectivity is always necessary before MS analysis. In this study, we developed a phosphorylated cotton fiber-based Ti(IV)-IMAC material (termed as: Cotton Ti-IMAC) that can serve as a novel platform for phosphopeptide enrichment. The cotton fiber can be effectively grafted with phosphate groups in a single step, where the titanium ions can then be immobilized onto to capture phosphopeptides. The material can be prepared with cost-effective reagents within only 4 hours. Benefiting from the flexibility and filterability of cotton fibers, the material can be easily packed as a spin-tip and make the enrichment process more convenient. Cotton Ti-IMAC successfully enriched phosphopeptides from protein standard digests and exhibited a high selectivity (β-casein/BSA = 1:1000) and excellent sensitivity (0.1 fmol/µL). Moreover, 2354 phosphopeptides was identified in a single LC-MS/MS injection after enriching from only 100 µg HeLa cell digests, with an enrichment specificity up to 97.51%. Taken together, we believe Cotton Ti-IMAC is ready to serve as a widely applicable and robust platform for achieving large-scale phosphopeptide enrichment and expanding our knowledge of phosphoproteomics in complex biological systems.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Danqing Wang  

LAB HEAD: Lingjun Li

PROVIDER: PXD037549 | Pride | 2023-10-23

REPOSITORIES: Pride