QconCAT

What is a QconCAT?

A QconCAT (Quantification conCATamer) is an artificial protein, generated by concatenation of proteotypic peptides.

Advantages

  • Accurate quantification
  • Easy multiplexing (quantify a complete proteome using multiple QconCATs)
  • Scalable to 1000’s of samples/data points
  • Reproducible
  • Robust synthesis of large amounts
  • Easy integration into established quantitative protein analysis setup
  • Compatible with other methods
  • Internal sample preparation control

Application areas

QconCATs are valuable tools for research applications involving quantitative proteomics and have been used in numerous academic and industrial research projects.

Large scale quantification

A single custom-made QconCAT enables targeted measurement and quantification of several tens of proteins. Using multiple QconCATs facilitates quantification of hundreds of proteins in a single experiment.

QconCATs by PolyQuant were used for absolute quantification of complete proteomes, networks and pathways.

Selected publications

  • Direct and Absolute Quantification of over 1800 Yeast Proteins via Selected Reaction Monitoring.
    Lawless C, Holman SW, Brownridge P, Lanthaler K, Harman VM, Watkins R, Hammond DE, Miller RL, Sims PF, Grant CM, Eyers CE, Beynon RJ, Hubbard SJ.
    Mol Cell Proteomics. 2016 Apr;15(4):1309-22. [PubMed]
  • Absolute protein quantification of the yeast chaperome under conditions of heat shock.
    Mackenzie RJ, Lawless C, Holman SW, Lanthaler K, Beynon RJ, Grant CM, Hubbard SJ, Eyers CE.
    Proteomics. 2016 Jun 2; [PubMed]
  • Quantification of the proteins of the bacterial ribosome using QconCAT technology.
    Al-Majdoub ZM, Carroll KM, Gaskell SJ, Barber J.
    J Proteome Res. 2014 Mar 7;13(3):1211-22. [PubMed]
  • Quantitative analysis of chaperone network throughput in budding yeast.
    Brownridge P, Lawless C, Payapilly AB, Lanthaler K, Holman SW, Harman VM, Grant CM, Beynon RJ, Hubbard SJ.
    Proteomics. 2013 Apr;13(8):1276-91. [PubMed]
  • A mass spectrometric strategy for absolute quantification of Plasmodium falciparum proteins of low abundance.
    Southworth PM, Hyde JE, Sims PF.
    Malar J.;2011 Oct 25;10:315. [PubMed]

Complex stoichiometry and isoform specificity

QconCATs are perfectly suited for absolute quantification of components of protein complexes, either in whole cell lysates or after immunoprecipitation. By selecting isoform-specific tryptic peptides, QconCATs allow quantification of highly homologous proteins that can otherwise not be distinguished by antibodies.

Selected publications

  • PIKES Analysis Reveals Response to Degraders and Key Regulatory Mechanisms of the CRL4 Network.
    Reichermeier KM, Straube R, Reitsma JM, Sweredoski MJ, Rose CM, Moradian A, den Besten W, Hinkle T, Verschueren E, Petzold G, Thomä NH, Wertz IE, Deshaies RJ, Kirkpatrick DS.
    Mol Cell. 2020 Jan 15.[Pubmed]
  • Stoichiometry, Absolute Abundance, and Localization of Proteins in the Bacillus cereus Spore Coat Insoluble Fraction Determined Using a QconCAT Approach.
    Stelder SK, Benito de Moya C, Hoefsloot HCJ, de Koning LJ, Brul S, de Koster CG.
    J Proteome Res. 2018 Feb 2;17(2):903-917 [Pubmed]
  • Rigorous determination of the stoichiometry of protein phosphorylation using mass spectrometry.
    Johnson H, Eyers CE, Eyers PA, Beynon RJ, Gaskell SJ.
    J Am Soc Mass Spectrom. 2009 Dec;20(12):2211-20. [PubMed]
  • COMMD Family Regulates Plasma LDL Levels and Attenuates Atherosclerosis Through Stabilizing the CCC Complex in Endosomal LDLR Trafficking.
    Fedoseienko A, Wijers M, Wolters JC, Dekker D, Smit M, Huijkman N, Kloosterhuis N, Klug H, Schepers A, Willems van Dijk K, Levels JH, Billadeau DD, Hofker MH, van Deursen J, Westerterp M, Burstein E, Kuivenhoven JA, van de Sluis B.
    Circ Res. 2018 Mar 15 [Pubmed]
  • Characterization of CYP2B6 K262R allelic variants by quantitative allele-specific proteomics using a QconCAT standard
    Barber J, Russell MR, Rostami-Hodjegan A, Achour B.
    J Pharm Biomed Anal. 2020 Jan 30;178:112901. [Pubmed]

Watch this video by Rob Beynon explaining how protein isoforms can be quantified using QconCAT technology:

show video

Biomarker discovery and validation, clinical research and diagnostics

QconCATs enhance data quality of targeted measurements in biomarker research projects. In clinical research, QconCATs can be also applied to pharmacological analyses, vaccine development and diagnostics.

Selected publications

  • Targeted LC-MS/MS for the evaluation of proteomics biomarkers in the blood of neonates with necrotizing enterocolitis and late-onset sepsis.
    Chatziioannou AC, Wolters JC, Sarafidis K, Thomaidou A, Agakidis C, Govorukhina N, Kuivenhoven JA, Bischoff R, Theodoridis G.
    Anal Bioanal Chem. 2018 Nov;410(27):7163-7175. [Pubmed]
  • Non-uniformity of Changes in Drug-Metabolizing Enzymes and Transporters in Liver Cirrhosis: Implications for Drug Dosage Adjustment.
    El-Khateeb E, Achour B, Al-Majdoub ZM, Barber J, Rostami-Hodjegan A.
    Mol Pharm. 2021 Sep 6;18(9):3563-3577.[Pubmed]
  • Design and expression of a QconCAT protein to validate Hi3 protein quantification of influenza vaccine antigens.
    Smith DG, Gingras G, Aubin Y, Cyr TD.
    J Proteomics. 2016 Sep 2;146:133-40. [Pubmed]
  • Cov-MS: A Community-Based Template Assay for Mass-Spectrometry-Based Protein Detection in SARS-CoV-2 Patients
    Bart Van Puyvelde, Katleen Van Uytfanghe, Olivier Tytgat, Laurence Van Oudenhove, Ralf Gabriels, Robbin Bouwmeester, Simon Daled, Tim Van Den Bossche, Pathmanaban Ramasamy, Sigrid Verhelst, Laura De Clerck, Laura Corveleyn, Sander Willems, Nathan Debunne, Evelien Wynendaele, Bart De Spiegeleer, Peter Judak, Kris Roels, Laurie De Wilde, Peter Van Eenoo, Tim Reyns, Marc Cherlet, Emmie Dumont, Griet Debyser, Ruben t’Kindt, Koen Sandra, Surya Gupta, Nicolas Drouin, Amy Harms, Thomas Hankemeier, Donald J. L. Jones, Pankaj Gupta, Dan Lane, Catherine S. Lane, Said El Ouadi, Jean-Baptiste Vincendet, Nick Morrice, Stuart Oehrle, Nikunj Tanna, Steve Silvester, Sally Hannam, Florian C. Sigloch, Andrea Bhangu-Uhlmann, Jan Claereboudt, N. Leigh Anderson, Morteza Razavi, Sven Degroeve, Lize Cuypers, Christophe Stove, Katrien Lagrou, Geert A. Martens, Dieter Deforce, Lennart Martens, Johannes P. C. Vissers, and Maarten Dhaenens.
    JACS Au 2021, 1, 6, 750–765.[Pubmed]

Quality control

Production of therapeutic proteins in biological systems demands for sophisticated quality control as even low levels of process-related impurities (host cell proteins, HCP) negatively impact stability and efficacy of the biopharmaceutical products and may cause a potential risk for patient health. Determination and monitoring of the levels of HCPs using custom-made QconCATs, developed for a biopharmaceutical product, provides critical information for determination of protein quality and process evaluation.

more…

Most recent publications employing QconCAT technology
  • Modeling Phenotypic Heterogeneity of Glycogen Storage Disease Type 1a Liver Disease in Mice by Somatic CRISPR/CRISPR-associated protein 9– Mediated Gene Editing
    Rutten MGS, Derks TGJ, Huijkman NCA, Bos T, Kloosterhuis NJ, van de Kolk KCWA, Wolters JC, Koster MH, Bongiovanni J, Thomas RE, de Bruin A, van de Sluis B, and Oosterveer MH.
    HEPATOLOGY, VOL. 74, NO. 5, 2021[Pubmed]
  • Proteomics of Colorectal Cancer Liver Metastasis: a Quantitative Focus on Drug Elimination and Pharmacodynamics Effects
    Vasilogianni AM, Al-Majdoub ZM, Achour B, Peters SA, Rostami-Hodjegan A, Barber J.
    Br J Clin Pharmacol. 2021 Oct 1.[Pubmed]
  • The hepatocyte IKK:NF-kB axis promotes liver steatosis by stimulating de novo lipogenesis and cholesterol synthesis
    Heida A, Gruben N, Catrysse L, Koehorst M, Koster M, Kloosterhuis NJ, Gerding A, Havinga R, Bloks VM, Bongiovanni L, Wolters JC, van Dijk T, van Loo G, de Bruin A, Kuipers F, Koonen DPY, van de Sluis B.
    MOLECULAR METABOLISM 54 (2021)[Pubmed]
  • Non-uniformity of Changes in Drug-Metabolizing Enzymes and Transporters in Liver Cirrhosis: Implications for Drug Dosage Adjustment.
    El-Khateeb E, Achour B, Al-Majdoub ZM, Barber J, Rostami-Hodjegan A.
    Mol Pharm. 2021 Sep 6;18(9):3563-3577.[Pubmed]
  • Age-related susceptibility to insulin resistance arises from a combination of CPT1B decline and lipid overload
    Vieira-Lara MA, Dommerholt MB, Zhang W, Blankestijn M, Wolters JC, Abegaz F, Gerding A, van der Veen YT, Thomas R, van Os RP, Reijngoud DJ, Jonker JW, Kruit JK and Bakker BM.
    BMC Biology (2021) 19:154.[Pubmed]
  • Pipelines and Systems for Threshold-Avoiding Quantification of LC−MS/MS Data
    Sánchez Brotons A, Eriksson JO, Kwiatkowski M, Wolters JC, Kema IP, Barcaru A, Kuipers F, Bakker SJL, Bischoff R, Suits F, and Horvatovich P.
    Anal. Chem. 2021, 93, 11215−11224.[Pubmed]
  • Impaired Very-Low-Density Lipoprotein catabolism links hypoglycemia to hypertriglyceridemia in Glycogen Storage Disease type Ia
    Hoogerland JA, Peeks F, Hijmans BS, Wolters JC, Kooijman S, Bos T, Bleeker A, van Dijk TH, Wolters H, Gerding A, van Eunen K, Havinga R, Pronk ACM, Rensen PCN, Mithieux G, Rajas F, Kuipers F, Reijngoud DJ, Derks TGJ, Oosterveer MH.
    J Inherit Metab Dis. 2021;44:879–892.[Pubmed]
  • Cholangiopathy and Biliary Fibrosis in Cyp2c70-Deficient Mice Are Fully Reversed by Ursodeoxycholic Acid
    de Boer JF, de Vries HD, Palmiotti A, Li R, Doestzada M, Hoogerland JA, Fu J, La Rose AM, Westerterp M, Mulder NL, Hovingh MV, Koehorst M, Kloosterhuis NJ, Wolters JC, Bloks VW, Haas JT, Dombrowicz D, Staels B, van de Sluis B, Kuipers F.
    Cell Mol Gastroenterol Hepatol 2021;11:1045–1069.[Pubmed]
  • Proteomic Quantification of Changes in Abundance of Drug-Metabolizing Enzymes and Drug Transporters in Human Liver Cirrhosis: Different Methods, Similar Outcomes
    El-Khateeb E, Al-Majdoub ZM, Rostami-Hodjegan A, Barber J, Achour B
    Drug Metabolism and Disposition May 27, 2021.[Pubmed]
  • Cov-MS: A Community-Based Template Assay for Mass-Spectrometry-Based Protein Detection in SARS-CoV-2 Patients
    Van Puyvelde B, Van Uytfanghe K, Tytgat O, Van Oudenhove L, Gabriels R, Bouwmeester R, Daled S, Van Den Bossche T, Ramasamy P, Verhelst S, De Clerck L, Corveleyn L, Willems S, Debunne N, Wynendaele E, De Spiegeleer B, Judak P, Roels K, De Wilde L, Van Eenoo P, Reyns T, Cherlet M, Dumont E, Debyser G, t’Kindt R, Sandra K, Gupta S, Drouin N, Harms A, Hankemeier T, Jones DJL, Gupta P, Lane D, Lane CS, El Ouadi S, Vincendet JB, Morrice N, Oehrle S, Tanna N, Silvester S, Hannam S, Sigloch FC, Bhangu-Uhlmann A, Claereboudt J, Anderson NL, Razavi M, Degroeve S, Cuypers L, Stove C, Lagrou K, Martens GA, Deforce D, Martens L, Vissers JPC, Dhaenens M.
    JACS Au 2021, 1, 6, 750–765.[Pubmed]

 

Original publications introducing the QconCAT method

  • Beynon R. J., Doherty M. K., Pratt J. M., Gaskell S. J. Multiplexed absolute quantification in proteomics using artificial QCAT proteins of concatenated signature peptides.
    Nature Methods Vol. 2 (8), August 2005, 587-589. [download]
  • Pratt J. M., Simpson D. M., Doherty M. K., Rivers J., Gaskell S. J., Beynon R. J. Multiplexed absolute quantification for proteomics using concatenated signature peptides encoded by QconCAT genes.
    Nature Protocols Vol. 1 (2), 2006, 1029-1043. [download]

 

Contact us to find out more about the implementation of custom-made QconCATs in your research project or benefit from our experience by confiding us with a full service quantification project.

Kommentare sind geschlossen.