Systematic Evaluation of Immobilized Trypsin-Based Fast Protein Digestion for Deep and High-Throughput Bottom-Up Proteomics.

ABSTRACT: Immobilized trypsin (IM) has been recognized as an alternative to free trypsin (FT) for accelerating protein digestion 30 years ago. However, some questions of IM still need to be answered. How does the solid matrix of IM influence its preference for protein cleavage and how well can IM perform for deep bottom-up proteomics compared to FT? By analyzing Escherichia coli proteome samples digested with amine or carboxyl functionalized magnetic bead-based IM (IM-N or IM-C) or FT, it is observed that IM-N with the nearly neutral solid matrix, IM-C with the negatively charged solid matrix, and FT have similar cleavage preference considering the microenvironment surrounding the cleavage sites. IM-N (15 min) and FT (12 h) both approach 9000 protein identifications (IDs) from a mouse brain proteome

SUBMITTER: Shen X 

PROVIDER: S-EPMC5932237 | biostudies-literature | 2018 May

REPOSITORIES: biostudies-literature