Protocol

Analysis of the Topology of Protein Complexes Using Cross-Linking and Mass Spectrometry

This protocol was adapted from "Characterization of Protein Complexes," Chapter 10, in Proteins and Proteomics (ed. Simpson). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2003.

INTRODUCTION

This protocol is designed to allow identification of spatial relationships between proteins within a wide variety of multiprotein complexes. Details for optimizing cross-linking of proteins within multiprotein complexes are provided. Following the cross-linking reaction, the proteins are separated by one-dimensional PAGE, the cross-linked proteins are isolated from the gel, and the individual members of the cross-linked complexes are identified by mass spectrometry. The protocol can be used to analyze many protein complexes isolated by any purification technique, provided the protein complexes remain in their native configuration.