Proteome-wide Alkylation to Improve Peptide Sequence Coverage and Detection Sensitivity

Effective ionization of peptides is critical for the sensitivity and accuracy of proteomic analyses, especially in nanoproteomics and single-cell proteomics, where the material is scarce. Previous studies have demonstrated the potential of peptide alkylation to improve ionization on certain peptides. In this work, we extend the scope of peptide alkylation by systematically modifying peptides with alkyl chains ranging from one to six carbons, aiming to enhance the ionization signal across a broad peptide spectrum. Our global alkylation approach revealed a significant increase in the ionization signal for a large portion of peptides, underscoring the technique's potential to improve proteomic analyses' sensitivity and overall sequence coverage. By employing machine learning to examine the relationship between alkylation-induced signal enhancement and various peptide characteristics, we found that the increase in signal strength is closely connected to peptide hydrophobicity. This relationship suggests that alkylation can particularly augment the detectability of hydrophilic peptides. Our findings not only highlight the importance of hydrophobicity in peptide ionization efficiency but also suggest that strategic alkylation could be a powerful tool to overcome the limitations imposed by sample size in proteomic with limited sample size.