Absolute multiplexed protein quantification using QconCAT technology.
In addition to protein identification, protein quantification is becoming a key output of proteomic experiments. Although relative quantification techniques are more commonplace and central to discovery proteomics, most assays require absolute quantification. The growth in systems biology has also increased the demand for absolute protein abundance values for input into models. QconCATs are created by concatenating peptide sequences taken from the target proteins into artificial proteins. The QconCAT acts as a source of internal standards and enables parallel absolute quantification of multiple proteins. QconCATs are typically applied in targeted proteomic workflows and so benefit from the greater sensitivity and wider dynamic range of these approaches. In this chapter, we discuss the design, construction, expression, and deployment of a QconCAT and the resulting experiments required for multiplex absolute quantification.
Protein Function Group, Institute of Integrative Biology, University of Liverpool, Liverpool, UK.
Philip J
PJ
Brownridge
Victoria M
VM
Harman
Deborah M
DM
Simpson
Robert J
RJ
Beynon
D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D002479:Inclusion Bodies / Q000737:chemistry; D007553:Isotope Labeling; D013058:Mass Spectrometry / Q000592:standards*; D010446:Peptide Fragments; D059748:Proteolysis; D040901:Proteomics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D012015:Reference Standards
D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins