Intramolecular cross‐linking experiments on cytochrome c and ribonuclease A using an isotope multiplet method

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Abstract
Mass spectral analysis of tryptic digests of cross‐linked proteins offers considerable promise as a simple technique to probe protein structure and study protein‐protein interactions. We describe the use of a 1:1 mixture of isotopically labeled and unlabeled cross‐linkers, disuccinimidyladipate (DSA) and dimethyladipimidate (DMA), to enhance visualization of cross‐linked peptides in a tryptic digest. Optimized intramolecular reactions of cytochrome c and ribonuclease A (RNase A) with DSA yielded an average of two cross‐links per protein molecule. After digestion of the cross‐linked cytochrome c with trypsin and analysis by liquid chromatography/mass spectrometry (LC/MS) and matrix‐assisted laser desorption/ionization (MALDI), eight modified peptides, five cross‐linked and two end‐capped, were detected by virtue of their doublet character. An eighth modified peptide's identity remained ambiguous because of its inability to fragment. The lysine‐lysine distance constraints obtained are discussed in the context of the known NMR and X‐ray structures of cytochrome c. Analysis of cross‐linked RNase A by LC/MS and MALDI yielded nine modified peptides, four of which were modified twice, as indicated by the isotopic triplets. Although seven of these peptides contained cross‐links, few distance constraints were gained due to the fact that the cross‐linked products were variations of modification of the same three lysine residues. Published in 2001 by John Wiley & Sons, Ltd.

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