Multi-Enzymatic Limited Digestion - the Next-Generation Sequencing for Proteomics

An innovative approach merging both the bottom-up and middle-down ideologies to provide fast, robust and comprehensive solutions for proteomics studies. The methodology, referred to as MELD for Multiple-Enzymatic Limited Digestion, capitalizes on a single two-hour long proteolytic step involving the synergic action of a diluted mix of several enzymes. While the multi-enzymatic benefits are retained, the lower enzyme concentrations induce a limitation in the proteolytic reaction. This feature engenders numerous missed cleavage events and results in abundant peptides of diverse length with overlapping stretches of residues. This outcome eventually increases the extent and confidence in the sequence coverage while opening roads for refined and comprehensive protein characterization. We here use Adalimumab, a therapeutic ~144 kDa human immunoglobulin G IgG1 as a candidate molecule to compare the respective efficacy of state-of-the-art complete mono-enzymatic strategies encompassed in individual or combinational workflows with the standard MELD protocol for extensive protein characterization. The benchmarking analysis was performed over 12 Adalimumab samples issued from independent proteolytic digestions: 6 samples were processed using the MELD methodology, 3 with the combinatory methodology and 3 with the mono-enzymatic tryptic methodology. They were all subjected to independent database searches, using both constrained and unconstrained cleavage site rules for the latter.