Discovering Protein–Protein Interactions using Co-Fractionation-Mass Spectrometry with Label-Free Quantitation

Abstract

Proteins generally achieve their functions through interactions with other proteins, so being able to determine which proteins interact with which other proteins underlies much of molecular biology. Co-fractionation (CF) is a mass spectrometry-based method for detecting proteome-wide protein–protein interactions. An attractive feature of CF is that it is not necessary to label or otherwise alter samples. Although we have previously published a widely used protocol for a label-incorporated CF methodology, no published protocols currently exist for the label-free variation. In this chapter, we describe a label-free CF-MS protocol. This protocol takes a minimum of a week, excluding the time for cell/tissue culture. It begins with cell/tissue lysis under non-denaturing conditions, after which intact protein complexes are isolated using size exclusion chromatography (SEC) where they are fractionated according to size. The proteins in each fraction are then prepared for mass spectrometry analysis where the constituent proteins are identified and quantified. Finally, we describe an in-house bioinformatics pipeline, PrInCE, to accurately predict protein complexes. Taken together, co-fractionation methodologies combined with mass spectrometry can identify and quantify thousands of protein–protein interactions in biological systems.