Protocol

Direct Enzymatic Digestion of Protein Complexes for MS Analysis

This protocol was adapted from "Identification of Novel Protein Complexes and Protein-Protein Interactions by Mass Spectrometry," Chapter 18, in Protein-Protein Interactions, 2nd ed. (eds. Golemis and Adams). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2005.

INTRODUCTION

The individual components of a purified protein complex can be determined by MS. Components can be separated by SDS-PAGE and subsequently digested, or the entire protein complex can be digested by chemical agents or by proteases. The mixture of peptides that results from digestion without prior separation is more complex than that obtained from the digestion of a single protein, and can best be resolved subsequently using HPLC in combination with a biphasic column. Protein complexes can be digested directly using several different protocols. Common methods include a double digest with endoproteinase Lys-C and trypsin, or a triple digest with elastase, subtilisin, and trypsin. Endoproteinase Lys-C cleaves carboxy-terminal to lysine residues, while trypsin cleaves carboxy-terminal to lysine and arginine residues. The triple digest uses the relatively nonspecific proteases elastase and subtilisin to create a large number of overlapping peptides for the analysis of post-translational modifications. The double and triple digest protocols begin with the reduction and alkylation of cysteine residues to disrupt disulfide bonds and, therefore, higher-order protein structure.