Investigation of Tryptic Protein Digestion in Microdroplets and in Bulk Solution

Abstract

Recent studies have shown that ultrafast enzymatic digestion of proteins can be achieved in microdroplet within 250 μs. 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 β-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.

Influence of particle z‐potential and experimental procedure on protein corona formation and multicomponent aggregation

“…Data‐dependent acquisition (DDA) MS/MS was performed where the first event was the survey positive mass scan (m/z range of 250–1800), followed by 20 higher‐energy C‐trap dissociation (HCD) events (30% normalized collision energy, NCE) on the 20 most abundant ions detected from the first event. Resolution for MS/MS analysis was set to 70,000 (AGC target of 3E6) and 17,500 (AGC target of 5E4) for survey scans and (HCD) scans 26 . Mass spectra was recorded using Thermo Xcalibur, and raw data were analyzed using Thermo Proteome Discoverer (PD) 2.4.…”

“…Resolution for MS/MS analysis was set to 70,000 (AGC target of 3E6) and 17,500 (AGC target of 5E4) for survey scans and (HCD) scans. 26 Mass spectra was recorded using Thermo Xcalibur, and raw data were analyzed using Thermo Proteome Discoverer (PD) 2.4. A database FASTA file was generated from Uniport under a search of bovine plasma (33,000+ entries).…”

Rapid Characterization of Antibodies via Automated Flow Injection Coupled with Online Microdroplet Reactions and Native-pH Mass Spectrometry

“…Recently, we have reported an ultrafast enzymatic digestion (<1 ms) of various proteins in microdroplets in which antibody light and heavy chains were shown to be digested by trypsin in less than 1 ms. , Ultrafast digestion of intact antibody was also demonstrated . However, most of the antibody digestion experiments involved offline MS analysis of the collected microdroplets resulting from spraying a manually mixed antibody and enzyme solution, making the total analysis time over 20 min.…”

“Lab of the Future”─Today: Fully Automated System for High-Throughput Mass Spectrometry Analysis of Biotherapeutics

“…Commonly used mass spectrometry (MS) methods for biologics include intact and subunit mass analysis and peptide mapping. − The increasing complexity of new therapeutic modalities is now creating a demand for more diverse MS techniques to characterize molecules. These new MS techniques employ a wide variety of wet chemistry methods, − high-throughput sample introduction, − separation in-line with MS instrumentation, − higher-order structural characterization, , and data analysis tools. However, MS is a time-consuming technique that requires tedious sample preparation, data acquisition, and data analysis.…”