Investigation of Tryptic Protein Digestion in Microdroplets and in Bulk Solution

Recent studies have shown that ultrafast enzymatic digestion of proteins can be achieved in microdroplet within 250 mu s. Further investigation of peptides resulting from microdroplet digestion (MD) would be necessary to evaluate it as an alternative to the conventional bulk digestion for bottom-up and biotherapeutic protein characterization. Herein we examined and compared protein tryptic digestion in both MD and bulk solution. In the case of MD of beta-lactoglobulin B, the preservation of long peptides was observed due to the short digestion time. In addition, MD is applicable to digest both high- and low-abundance proteins in mixture. In the case of digesting NIST 8671 mAb antibody containing a low level of commonly encountered host cell protein (HCP) PLBL2 (mAb:PLBL2 = 100:1 by weight), MD produced lower levels of digestion-induced chemical modifications of asparagine/glutamine deamidation, compared with overnight digestion. No significant difference between MD and bulk digestion was observed in terms of trypsin digestion specificity based on examination of semi- and unspecific-cleaved peptides. Our study suggests that MD, a fast digestion approach, could be adopted for bottom-up proteomics research and for peptide mapping of mAbs to characterize site-specific deamidation and glycosylation, for the purpose of development of biopharmaceuticals.

Automated summary

Recent studies have shown that ultrafast enzymatic digestion of proteins can be achieved in microdroplets within 250 μs. This study compared protein tryptic digestion in both microdroplets and bulk solution, and found that microdroplet digestion preserved long peptides due to the short digestion time. Microdroplet digestion was also effective for digesting high- and low-abundance proteins in a mixture. Additionally, microdroplet digestion resulted in lower levels of digestion-induced chemical modifications compared to overnight digestion. This study suggests that microdroplet digestion could be a fast and effective method for bottom-up proteomics research and peptide mapping of monoclonal antibodies.