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

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. Compared to FT, IM-N has no bias in the digestion of proteins that are involved in various biological processes, are located in different components of cells, have diverse functions, and are expressed in varying abundance. A high-throughput bottom-up proteomics workflow comprising IM-N-based rapid protein cleavage and fast CZE-MS/MS enables the completion of protein sample preparation, CZE-MS/MS analysis, and data analysis in only 3 h, resulting in 1000 protein IDs from the mouse brain proteome.