The Application of a Hypothesis-driven Strategy to the Sensitive Detection and Location of Acetylated Lysine Residues

John R. Griffiths, Richard D. Unwin, Caroline A. Evans, Siân H. Leech, Bernard M. Corfe, Anthony D. Whetton

Research output: Contribution to journalArticlepeer-review

Abstract

The application of a hypothesis-driven method for the sensitive determination of lysine acetylation sites on enzymatically digested proteins is described. Comparative sensitivity tests were carried out using serial dilution of an acetylated bovine serum albumin (AcBSA) digest to assess the performance of a multiple reaction monitoring (MRM)-based approach as compared to a more conventional precursor scanning (PS) method. Both methods were capable of selectively detecting an acetylated peptide at the low femtomole level when spiked into a background of 500 fmol six-protein tryptic digest. The MRM approach was roughly tenfold more sensitive than precursor scanning with one acetylated peptide detected and sequenced at the level of 2 fmol on-column. The technique was subsequently applied to a gel-derived sample of cytokeratin-8 (CK8) shown to contain acetylated lysine residues by Western blot analysis. The strategy applied herein, termed MRM-initiated detection and sequencing (MIDAS), resulted in the facile identification of novel sites of acetylation on this protein. © 2007 American Society for Mass Spectrometry.
Original languageEnglish
Pages (from-to)1423-1428
Number of pages5
JournalJournal of the American Society for Mass Spectrometry
Volume18
Issue number8
DOIs
Publication statusPublished - Aug 2007

Keywords

  • Acetylation
  • Amino Acid Sequence
  • Animals
  • methods: Blotting, Western
  • Cattle
  • Chromatography, Liquid
  • Humans
  • chemistry: Keratin-8
  • analysis: Lysine
  • Mass Spectrometry
  • chemistry: Serum Albumin, Bovine
  • metabolism: Trypsin

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