Detergent-Assisted Protein Digestion-On the Way to Avoid the Key Bottleneck of Shotgun Bottom-Up Proteomics.

Abstract

Gel-free bottom-up shotgun proteomics is the principal methodological platform for the state-of-the-art proteome research. This methodology assumes quantitative isolation of the total protein fraction from a complex biological sample, its limited proteolysis with site-specific proteases, analysis of the resulted peptides with nanoscaled reversed-phase high-performance liquid chromatography-(tandem) mass spectrometry (nanoRP-HPLC-MS and MS/MS), protein identification by sequence database search and peptide-based quantitative analysis. The most critical steps of this workflow are protein reconstitution and digestion; therefore, detergents and chaotropic agents are strongly mandatory to ensure complete solubilization of complex protein isolates and to achieve accessibility of all protease cleavage sites. However, detergents are incompatible with both RP separation and electrospray ionization (ESI). Therefore, to make LC-MS analysis possible, several strategies were implemented in the shotgun proteomics workflow. These techniques rely either on enzymatic digestion in centrifugal filters with subsequent evacuation of the detergent, or employment of MS-compatible surfactants, which can be degraded upon the digestion. In this review we comprehensively address all currently available strategies for the detergent-assisted proteolysis in respect of their relative efficiency when applied to different biological matrices. We critically discuss the current progress and the further perspectives of these technologies in the context of its advances and gaps.