고재윤 (가천대학교)

이후근 (가천대학교)

고재윤 (가천대학교)

이후근 (가천대학교)

양원준 (가천대학교)

 In bottom-up proteomics, enzymatic digestion influences protein identification and sequence coverage. Although in-gel, in-solution, filter-aided sample preparation (FASP), and S-Trap digestion have been developed, a single digestion step may not recover all peptide regions because of protein structure, limited accessibility, and hydrophobic interactions. FASP enables purification, buffer exchange, and digestion on a filter membrane, but material may remain after primary digestion.

In this study, the filter-retained fraction was evaluated for additional peptide recovery using methanol-assisted secondary digestion. Human serum proteins were digested using a FASP-based workflow, and the filter-retained fraction was treated with methanol and trypsin. Each condition was performed in triplicate, and LC-MS/MS was used to evaluate peptide and protein identification and coverage changes versus primary digestion.

Methanol-treated conditions (10–50%) yielded additional peptides absent from primary digestion. 10% methanol showed the highest proportion, corresponding to 6.8% of peptides identified in primary digestion. Among commonly identified proteins, 72 remained after excluding contaminants and ambiguous matches; 9 (12.5%) showed a coverage increase of at least 10 percentage points.