Quenching Trypsin Is Unnecessary in Filter-Based Bottom-Up Proteomics.

Abstract

Quenching digestions in proteomics prior to analysis is routine in order to eliminate residual protease activity. Residual activity leads to overdigestion, nonspecific star-activity, and back-exchange in isotopic ¹⁸O quantitation. Chemical and isobaric labeling (e.g., TMT/iTRAQ) of proteins or peptides for mass spectrometry-based proteomics is generally incompatible with ubiquitous postdigestion acidification. This necessitates buffer exchange and pH adjustments. We demonstrate that quenching is unnecessary with peptides generated from protein filter-traps, as trypsin activity and intact trypsin are negligible in the eluate from these preparations. Labeling can be directly performed on enzymatic digests from these methods, improving recovery, throughput, and ease of automation.