Stable isotope labelling in vivo as an aid to protein identification in peptide mass fingerprinting.
Peptide mass fingerprinting (PMF) is a powerful technique for identification of proteins derived from in-gel digests by virtue of their matrix-assisted laser desorption/ionization-time of flight mass spectra. However, there are circumstances where the under-representation of peptides in the mass spectrum and the complexity of the source proteome mean that PMF is inadequate as an identification tool. In this paper, we show that identification is substantially enhanced by inclusion of composition data for a single amino acid. Labelling in vivo with a stable isotope labelled amino acid (in this paper, decadeuterated leucine) identifies the number of such amino acids in each digest fragment, and show a considerable gain in the ability of PMF to identify the parent protein. The method is tolerant to the extent of labelling, and as such, may be applicable to a range of single cell systems.
Pratt
Julie M
JM
Department of Veterinary Preclinical Sciences, University of Liverpool, Liverpool, UK.
Robertson
Duncan H L
DH
Gaskell
Simon J
SJ
Riba-Garcia
Isabel
I
Hubbard
Simon J
SJ
Sidhu
Khushwant
K
Oliver
Stephen G
SG
Butler
Philip
P
Hayes
Andrew
A
Petty
June
J
Beynon
Robert J
RJ
D003903:Deuterium; D007930:Leucine / Q000032:analysis; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010449:Peptide Mapping / Q000379:methods*; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification*; D020543:Proteome / Q000737:chemistry / Q000302:isolation & purification; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000302:isolation & purification; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D010446:Peptide Fragments; D011506:Proteins; D020543:Proteome; D029701:Saccharomyces cerevisiae Proteins; D003903:Deuterium; D007930:Leucine