Ten years of QconCATs: Application of multiplexed quantification to small medically relevant proteomes

Abstract

In this report, we briefly review the application of Beynon and Gaskell's QconCAT methodology to the quantification of a variety of proteomes. We then describe the development of two QconCATs, intended for the analysis of small proteomes relevant to human medicine: the bacterial ribosome (30SCAT) and the human drug-metabolizing enzymes (cytochrome P450 and uridine 5′-diphospho-glucuronosyltransferase enzymes: MetCAT). The design of both QconCATs was supported by experimental identification of proteotypic peptides, leading to redundancy of information during quantification, and providing good evidence for complete digestion of both analyte and QconCAT. Both QconCATs are designed to be used with sequential LysC and trypsin digestion, and in the case of the 30SCAT, two LC MS/MS measurements are made, one following each digestion step. The MetCAT initially failed to express; successful expression was achieved in two ways: by a reshuffle of the order of the peptides, and by fusion of the original construct with the 30SCAT gene. Both QconCATs have been used to quantify samples of importance in human medicine.