Determination of protein contacts by chemical cross-linking with EDC and mass spectrometry

This unit describes a method for analyzing protein complexes by chemically cross-linking closely positioned amino acids. The cross-linked products are isolated by SDS-PAGE and the bands of interest are excised. Proteins in the excised gel piece are digested with trypsin and the resulting peptides recovered. Chemically bonded peptides are distinct from the multitude of unmodified single peptides by the presence of two carboxy-terminal ends. The protocol also describes incorporation of (18)O at the carboxy-terminal ends and purification and preparation of the peptide mixture for mass spectroscopy.