김선민 (충남대학교 대학원)

김선민 (충남대학교 대학원)

Phosphorylation is a key post-translational modification regulating diverse cellular processes, yet low phosphopeptide abundance necessitates efficient enrichment and fractionation. Although conventional fractionation methods provide high proteome coverage, they involve complex workflows and demand lengthy analysis times. We developed a streamlined phosphoproteomic workflow integrating IMAC-based enrichment with stepwise acetonitrile (ACN) gradient elution (10%, 25%, 50%) during desalting, enabling simultaneous enrichment and fractionation without additional equipment. We evaluated how stepwise ACN elution enhances phosphoproteomic coverage, and found that each fraction complementarily detected unique phosphopeptides, contributing to an overall improvement in coverage. Single-shot elution yielded 16,151 phosphopeptide MS2 spectra, whereas stepwise elution produced 16,144, 14,491, and 9,338 spectra in the 10%, 25%, and 50% fractions, indicating the 10% fraction alone matched single-shot depth while others added further depth. Whereas conventional enrichment efficiency assessment requires peptide identification, our in-house ID-Free evaluation method calculates the proportion of phosphopeptide-related spectra among total MS2 spectra, enabling rapid assessment of enrichment purity without identification. Using this method, the 10% ACN fraction was found to maintain high enrichment purity while providing phosphopeptide depth comparable to single-shot analysis, whereas the 25% and 50% ACN fractions further increased phosphopeptide depth at the expense of reduced purity and increased glycopeptide interference. These results support stepwise ACN elution as a practical, cost-effective strategy for deep phosphoproteomics, with ID-Free enabling rapid evaluation of enrichment efficiency and glyco-co-enrichment.​