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

Protein phosphorylation is an important
post-translational
modification
(PTM), which is involved in many important cellular functions. Understanding
protein phosphorylation at the molecular level is critical to deciphering
its relevant biological processes and signaling networks. Mass spectrometry
(MS) has become a powerful tool for the comprehensive profiling of
protein phosphorylation. Yet the low ionization efficiency and low
abundance of phosphopeptides among complex biological samples make
its MS analysis challenging; an enrichment strategy with high efficiency
and selectivity is always necessary prior to 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 covalently in a single step, where the
titanium ions can then be immobilized to enable capturing phosphopeptides.
The material can be prepared using cost-effective reagents within
only 4 h. Benefiting from the flexibility and filterability of cotton
fibers, the material can be easily packed as a spin-tip and make the
enrichment process convenient. Cotton Ti-IMAC successfully enriched
phosphopeptides from protein standard digests and exhibited a high
selectivity (BSA/β-casein = 1000:1) and excellent sensitivity
(0.1 fmol/μL). Moreover, 2354 phosphopeptides were profiled
in one LC-MS/MS injection after enriching from only 100 μg of
HeLa cell digests with an enrichment specificity of up to 97.51%.
Taken together, we believe that Cotton Ti-IMAC can serve as a widely
applicable and robust platform for achieving large-scale phosphopeptide
enrichment and expanding our knowledge of phosphoproteomics in complex
biological systems.