Partial acetylation of lysine residues improves intraprotein cross-linking

Authors

Xin Guo, University of California, San FranciscoFollow
Pradipta Bandyopadhyay, University of California, San Francisco
Birgit Schilling, Buck Institute for Age Research
Malin M. Young, Sandia National Laboratories
Naoaki Fujii, University of California, San Francisco
Tiba Aynechi, University of California, San Francisco
Richard Kiplin Guy, University of California, San Francisco
Irwin D. Kuntz, University of California, San Francisco
Bradford W. Gibson, Buck Institute for Age ResearchFollow

Document Type

Article

Publication Title

Analytical Chemistry

ISSN

0003-2700

Volume

80

Issue

4

DOI

10.1021/ac701636w

First Page

951

Last Page

960

Publication Date

1-18-2008

Abstract

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.

Recommended Citation

Guo, X., Bandyopadhyay, P., Schilling, B., Young, M. M., Fujii, N., Aynechi, T., Guy, R. K., Kuntz, I. D., & Gibson, B. W. (2008). Partial acetylation of lysine residues improves intraprotein cross-linking. Analytical Chemistry, 80(4), 951–960. DOI: 10.1021/ac701636w
https://scholarlycommons.pacific.edu/phs-facarticles/291