Sequence Coverage Optimization by HPLC Separation and Consecutive Enzymatic Digestion

摘要

Post Translational Modification Analysis often requires nearly 100 % protein sequence coverage. Peptides, resulting in enzymatic reactions, vary dramatically in the length, so their m/z values could be beyond the mass range of the conventional mass spectrometers. If second sequential digestion step with different enzyme is applied, too small fragments could be produced. To overcome such a problem, an additional enzymatic digestion step was carried out to break long tryptic peptides only into acceptable fragments. This method is based on good correlation between peptide size and its relative hydrophobicity index. Namely, long peptides have a trend to be more hydrophobic, and, consequently, have longer retention times.