title: DOSCATs: Double standards for protein quantification.
abstract: The two most common techniques for absolute protein quantification are based on either mass spectrometry (MS) or on immunochemical techniques, such as western blotting (WB). Western blotting is most often used for protein identification or relative quantification, but can also be deployed for absolute quantification if appropriate calibration standards are used. MS based techniques offer superior data quality and reproducibility, but WB offers greater sensitivity and accessibility to most researchers. It would be advantageous to apply both techniques for orthogonal quantification, but workflows rarely overlap. We describe DOSCATs (DOuble Standard conCATamers), novel calibration standards based on QconCAT technology, to unite these platforms. DOSCATs combine a series of epitope sequences concatenated with tryptic peptides in a single artificial protein to create internal tryptic peptide standards for MS as well as an intact protein bearing multiple linear epitopes. A DOSCAT protein was designed and constructed to quantify five proteins of the NF-κB pathway. For three target proteins, protein fold change and absolute copy per cell values measured by MS and WB were in excellent agreement. This demonstrates that DOSCATs can be used as multiplexed, dual purpose standards, readily deployed in a single workflow, supporting seamless quantitative transition from MS to WB.
authors.affiliation: Centre for Proteome Research, Institute of Integrative Biology, Biosciences Building, University of Liverpool, Liverpool L69 7ZB, UK.
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Bennett
authors.affiliation: Centre for Proteome Research, Institute of Integrative Biology, Biosciences Building, University of Liverpool, Liverpool L69 7ZB, UK.
authors.forename: Deborah M
authors.initials: DM
authors.lastname: Simpson
authors.affiliation: Centre for Proteome Research, Institute of Integrative Biology, Biosciences Building, University of Liverpool, Liverpool L69 7ZB, UK.
authors.forename: Stephen W
authors.initials: SW
authors.lastname: Holman
authors.affiliation: Institute of Ageing and Chronic Disease, The Apex Building, 6 West Derby St., Liverpool L7 8TX, UK.
authors.forename: Sheila
authors.initials: S
authors.lastname: Ryan
authors.affiliation: Centre for Proteome Research, Institute of Integrative Biology, Biosciences Building, University of Liverpool, Liverpool L69 7ZB, UK.
authors.forename: Philip
authors.initials: P
authors.lastname: Brownridge
authors.affiliation: Centre for Proteome Research, Institute of Integrative Biology, Biosciences Building, University of Liverpool, Liverpool L69 7ZB, UK.
authors.forename: Claire E
authors.initials: CE
authors.lastname: Eyers
authors.affiliation: Badrilla Ltd. Leeds Innovation Centre, School of Biomedical Sciences, University of Leeds, Leeds LS2 9JT, UK.
authors.forename: John
authors.initials: J
authors.lastname: Colyer
authors.affiliation: Centre for Proteome Research, Institute of Integrative Biology, Biosciences Building, University of Liverpool, Liverpool L69 7ZB, UK.
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Beynon
mesh_terms: D006801:Humans; D010446:Peptide Fragments / Q000032:analysis / Q000592:standards; D011506:Proteins / Q000032:analysis* / Q000592:standards*; D020543:Proteome / Q000032:analysis* / Q000592:standards*; D040901:Proteomics / Q000379:methods*; D012015:Reference Standards
chemical_list: D010446:Peptide Fragments; D011506:Proteins; D020543:Proteome