Intramolecular cross-linking experiments on cytochrome c and ribonuclease A using an isotope multiplet method.
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.
Pearson
Kara M
KM
National Heart, Lung, and Blood Institute, NIH, Bethesda, MD 20892, USA.
Pannell
Lewis K
LK
Fales
Henry M
HM
D003432:Cross-Linking Reagents; D003574:Cytochrome c Group / Q000737:chemistry*; D006868:Hydrolysis; D007202:Indicators and Reagents; D008958:Models, Molecular; D009211:Myoglobin / Q000737:chemistry; D010455:Peptides / Q000737:chemistry; D011487:Protein Conformation; D012259:Ribonuclease, Pancreatic / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin
D003432:Cross-Linking Reagents; D003574:Cytochrome c Group; D007202:Indicators and Reagents; D009211:Myoglobin; D010455:Peptides; D012259:Ribonuclease, Pancreatic; D014357:Trypsin