The application of a hypothesis-driven strategy to the sensitive detection and location of acetylated lysine residues.
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.
Griffiths
John R
JR
Stem Cell and Leukaemia Proteomics Laboratory, Division of Cancer Studies, University of Manchester, Manchester, United Kingdom.
Unwin
Richard D
RD
Evans
Caroline A
CA
Leech
Siân H
SH
Corfe
Bernard M
BM
Whetton
Anthony D
AD
D000107:Acetylation; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western / Q000379:methods*; D002417:Cattle; D002853:Chromatography, Liquid; D006801:Humans; D053533:Keratin-8 / Q000737:chemistry*; D008239:Lysine / Q000032:analysis / Q000737:chemistry*; D013058:Mass Spectrometry; D012710:Serum Albumin, Bovine / Q000737:chemistry*; D014357:Trypsin / Q000378:metabolism
D053533:Keratin-8; D012710:Serum Albumin, Bovine; D014357:Trypsin; D008239:Lysine