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

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.

Identification of cross-linked peptides from large sequence databases

“…Improvement in mass spectrometry (MS) technology has made this conceivable, in particular high mass precision spectrometers and the development of isotopically coded cross-linkers [3][4][5] . Several studies have proven the general feasibility of this approach 3,[6][7][8][9] . Nonetheless, this methodology has only been used for single proteins or small, purified protein complexes so far.…”

Covalent Adducts Arising from the Decomposition Products of Lipid Hydroperoxides in the Presence of Cytochrome c

“…Person, et al, previously reported adduction by both benzoquinone and glutathione-S-yl-1,4-benzoquinone at sites K 86 and K 87 on cytochrome C, suggesting this is a highly reactive region of the protein (23,24). Pearson, et al, in a study of protein crosslinking agents, also observed reaction at the corresponding locus on bovine cytochrome C, although they declined to speculate on the reasons for activity at these positions (25). Person and co-workers suggest that the enhanced reactivity may reflect conformational flexibility, since lysines 86 and 87 are in loop regions of the protein (23), but this would not explain why only DODE appears to react at these positions.…”

Strategy for selective chemical cross-linking of tyrosine and lysine residues

“…In a recent study by another group using proteolytic digests to localize the cross-links in cytochrome-c, only one cross-link was observed with lysine reactive crosslinkers when the reaction was optimized to yield a single cross-link per protein molecule [9]. At higher cross-linker concentration optimized to yield two crosslinks per protein molecule, the digest yielded only four additional cross-links.…”