Partial acetylation of lysine residues improves intraprotein cross-linking

Anal Chem. 2008 Feb 15;80(4):951-60. doi: 10.1021/ac701636w. Epub 2008 Jan 18.


Intramolecular cross-linking coupled with mass spectrometric identification of cross-linked amino acids is a rapid method for elucidating low-resolution protein tertiary structures or fold families. However, previous cross-linking studies on model proteins, such as cytochrome c and ribonuclease A, identified a limited number of peptide cross-links that are biased toward only a few of the potentially reactive lysine residues. Here, we report an approach to improve the diversity of intramolecular protein cross-linking starting with a systematic quantitation of the reactivity of lysine residues of a model protein, bovine cytochrome c. Relative lysine reactivities among the 18 lysine residues of cytochrome c were determined by the ratio of d0 and acetyl-d3 groups at each lysine after partial acetylation with sulfosuccinimidyl acetate followed by denaturation and quantitative acetylation of remaining unmodified lysines with acetic-d6 anhydride. These lysine reactivities were then compared with theoretically derived pKa and relative solvent accessibility surface values. To ascertain if partial N-acetylation of the most reactive lysine residues prior to cross-linking can redirect and increase the observable Lys-Lys cross-links, partially acetylated bovine cytochrome c was cross-linked with the amine-specific, bis-functional reagent, bis(sulfosuccinimidyl)suberate. After proteolysis and mass spectrometry analysis, partial acetylation was shown to significantly increase the number of observable peptides containing Lys-Lys cross-links, shifting the pattern from the most reactive lysine residues to less reactive ones. More importantly, these additional cross-linked peptides contained novel Lys-Lys cross-link information not seen in the non-acetylated protein and provided additional distance constraints that were consistent with the crystal structure and facilitated the identification of the proper protein fold.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Acetates / chemistry
  • Acetylation
  • Amino Acid Sequence
  • Chromatography, Liquid / methods
  • Cross-Linking Reagents / chemistry*
  • Cytochromes c / chemistry
  • Lysine / chemistry*
  • Molecular Sequence Data
  • Peptides / chemistry
  • Protein Denaturation
  • Protein Folding
  • Protein Structure, Tertiary
  • Proteins / chemistry*
  • Ribonuclease, Pancreatic / chemistry
  • Solvents / chemistry
  • Succinimides / chemistry
  • Tandem Mass Spectrometry / methods


  • Acetates
  • Cross-Linking Reagents
  • Peptides
  • Proteins
  • Solvents
  • Succinimides
  • sulfosuccinimidyl acetate
  • Cytochromes c
  • Ribonuclease, Pancreatic
  • Lysine