Affinity Purification and Preparation of Peptides for Mass Spectrometry from C. elegans

Abstract

Affinity purification of a target protein followed by mass spectrometry of the purified peptides can be used to identify physical interactors of the protein of interest. Using this biochemical approach on proteins from whole organisms such as C. elegans can reveal novel in vivo protein interactions that cannot be identified using homology-based predictions or in vitro approaches. Here we describe affinity purification of a GFP-tagged target protein from whole worm lysates, digestion of the purified proteins into peptides, and preparation of the peptides for analysis by mass spectrometry. This protocol has been optimized for ChromoTek GFP-Trap® Magnetic Agarose beads, but it may be used with other tags and antibody-conjugated beads.