Direct Analysis and Identification of Proteins in Mixtures by LC/MS/MS and Database Searching at the Low-Femtomole Level

TLDR

The study develops a method for directly identifying proteins in mixtures using microcolumn reversed‑phase LC/MS/MS. Proteins are enzymatically digested into peptides, which are online separated and analyzed by tandem mass spectrometry to generate spectra that are searched against a protein database. The method reliably identifies proteins differing 30‑fold in abundance, reproducibly detects proteins at low femtomole levels, and enables rapid analysis of enriched protein complexes.

Abstract

A method to directly identify proteins contained in mixtures by microcolumn reversed-phase liquid chromatography electrospray ionization tandem mass spectrometry (LC/MS/MS) is studied. In this method, the mixture of proteins is digested with a proteolytic enzyme to produce a large collection of peptides. The complex peptide mixture is then separated on-line with a tandem mass spectrometer, acquiring large numbers of tandem mass spectra. The tandem mass spectra are then used to search a protein database to identify the proteins present. Results from standard protein mixtures show that proteins present in simple mixtures can be readily identified with a 30-fold difference in molar quantity, that the identifications are reproducible, and that proteins within the mixture can be identified at low femtomole levels. Based on these studies, methodology has been developed for direct LC/MS/MS analysis of proteins enriched by immunoaffinity precipitation, specific interaction with a protein−protein fusion product, and specific interaction with a macromolecular complex. The approach described in this article provides a rapid method for the direct identification of proteins in mixtures.