Qualitative and quantitative proteomics by two-dimensional gel electrophoresis, peptide mass fingerprint and a chemically-coded affinity tag (CCAT).
The chemically-coded affinity tag (CCAT) method combines standard electrophoresis protocols with MALDI-TOF-MS analysis to identify and quantify protein abundances in complex samples in one step. This method is designed to fit into the workflow of SDS-PAGE or two-dimensional electrophoresis (2-DE) only requiring basic proteome laboratory equipment. Prior to electrophoresis two protein samples are separately labelled with a heavy or a light version of the CCAT reagent via reduced cysteines in the proteins. Equal amounts are then combined and electrophoretically separated. Proteins can then be excised from the gel to obtain their peptide mass fingerprint by mass spectrometry. This fingerprint enabled not only identification, but also quantification by comparing relative peak intensities of CCAT-labelled peptides. In this article, we display how the CCAT method can be used to analyse two protein samples in one gel and that the peak intensities of labelled peptides reflect the abundance of a protein in it.
Watt
Steven Alexander
SA
Lehrstuhl für Genetik, Fakultät für Biologie, Universität Bielefeld, POB 100131, 33501 Bielefeld, Germany.
Patschkowski
Thomas
T
Kalinowski
Jörn
J
Niehaus
Karsten
K
D000345:Affinity Labels / Q000737:chemistry; D001426:Bacterial Proteins / Q000032:analysis* / Q000737:chemistry*; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D010449:Peptide Mapping / Q000379:methods*; D040901:Proteomics / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D016959:Xanthomonas campestris / Q000378:metabolism
D000345:Affinity Labels; D001426:Bacterial Proteins