QconCAT Technology

QconCAT Technology

Illustration-TechnologyQconCATs (Quantification conCATamer) are custom-made synthetic proteins, usually comprising of up to 50 heavy isotope-labelled proteotypic peptides, functioning as true internal standards for absolute protein quantification using mass spectrometry.

Original publications:
  • Beynon R. J. et al., Multiplexed absolute quantification in proteomics using artificial QCAT proteins of concatenated signature peptides. Nature Methods Vol. 2 (8), August 2005, 587-589.
  • Pratt J. M. et al., Multiplexed absolute quantification for proteomics using concatenated signature peptides encoded by QconCAT genes. Nature Protocols Vol. 1 (2), 2006, 1029-1043.
Illustration-TechnologyProteotypic peptides are selected using either available data or by in silico analysis. The QconCAT is then produced as heavy isotope-labelled synthetic protein.
Absolute protein quantification is performed by adding the QconCAT in known quantity to the sample (e. g. a cell extract).
Subsequent proteolysis, for example with trypsin, releases the proteotypic peptides both from the target proteins and the QconCAT for analysis in a mass spectrometer.
The intensity of the reference peptide peaks allows calculation of the exact absolute amount of each target protein.
Illustration-TechnologyQconCATs save time preparing peptide mixes by enabling spiking of the analyte sample with up to 50 reference peptides in a single pipetting step.
Besides that, the design of a QconCAT as artificial polypeptide has several other advantages compared to synthetic peptides:
  • Synthesize difficult peptides without limitation in peptide length
  • Include flanking amino acids to control for variations caused by missed cleavages
  • Prevent peptide loss through adsorption to vessel walls
  • Release the target peptides in a 1:1 ratio
  • Internal control for sample processing by early addition of the QconCAT during sample preparation
Illustration-TechnologyAbsolute protein quantification using QconCAT reference standards can be established on all commonly used mass spectrometry platforms. Our technology is compatible with other methods and can be integrated into established quantitative protein analysis workflows.

QconCATs are highly versatile tools for various research projects in academia and industry and have been successfully applied in exploratory studies as well as in high throughput analyses.
A list of selected references using QconCATs by PolyQuant can be found here.

Proteomics Quality control Host cell protein analysis Complex stoichiometry and isoform specificity Biomarker research Clinical proteomics Agricultural biotechnology Instrument calibration Food safety Cell physiology

Selected references

  • 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]
    Lawless et al. quantified over 1800 S. cerevisiae proteins by selected reaction monitoring (SRM) mass spectrometry using over 100 QconCATs.
  • 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]
    Mackenzie et al. determined copy per cell values for 49 key chaperones in S. cerevisiae under conditions of normal growth and heat shock, by selected reaction monitoring (SRM) mass spectrometry using QconCATs as reference peptides.
  • Full humanization of the glycolytic pathway in Saccharomyces cerevisiae
    Boonekamp FJ, Knibbe E, Vieira-Lara MA, Wijsman M, Luttik MAH, van Eunen K, Ridder MD, Bron R, Almonacid Suarez AM, van Rijn P, Wolters JC, Pabst M, Daran JM, Bakker BM, Daran-Lapujade P.
    Cell Rep. 2022 Jun 28;39(13):111010. [Pubmed]
    Boonekamp et al. generated and characterized a yeast strain with a humanized glycolytic pathway, providing a new model to study human glycolysis in a simplified context. In their study they used 6 QconCATs to determine absolute concentrations of glycolytic targets by targeted proteomics.
  • CRAF dimerization with ARAF regulates KRAS-driven tumor growth
    Venkatanarayan A, Liang J, Yen I, Shanahan F, Haley B, Phu L, Verschueren E, Hinkle TB, Kan D, Segal E, Long JE, Lima T, Liau NPD, Sudhamsu J, Li J, Klijn C, Piskol R, Junttila MR, Shaw AS, Merchant M, Chang MT, Kirkpatrick DS, Malek S.
    Cell Rep. 2022 Feb 8;38(6):110351. [PubMed]
    Venkatanarayan et al. used quantitative proteomics to demonstrate increased levels of CRAF:ARAF dimers in KRAS mutant cells and PRM analysis to quantify the stoichiometric relationships between wt and mutant MAPK components using a QconCAT incorporating wild type and mutant sequences for detection of disease associated mutations.
  • 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]
    Reichermeier et al. determined the stoichiometric relationships between approx. 30 proteins in cell lysates and immunoprecipitated samples using targeted proteomics and a QconCAT comprising of 67 peptides for 31 proteins.
  • 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]
    Stelder et al. quantified crucial spore proteins using a QconCAT reference standard, determining the absolute abundance of 21 proteins, covering approx. 5.66% of the total spore weight in wild type and approx. 4.13% in the ΔCotE mutant.
  • Rigorous determination of the stoichiometry of protein phosphorylation using mass spectrometry
    Johnson H, Eyers CE, Eyers PA, Beynon RJ, Gaskell SJ.
    Journal of the American Society for Mass Spectrometry 2009 Dec;20(12):2211-20.
    Johnson et al. use QconCATs to determine absolute protein concentrations and the stoichiometry of phosphorylation at predefined sites.
  • 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]
    Fedoseienko et al. used QconCATs to quantify the protein concentrations of the COMMDs, components of retromer, the CCC and WASH complexes, etc in the samples of the different mouse models.
  • 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]
    Barber et al. used a QconCAT standard for targeted proteomics, simultaneously determining protein abundance and missense polymorphisms.
  • Loss of hepatic SMLR1 causes hepatosteatosis and protects against atherosclerosis due to decreased hepatic VLDL secretion
    van Zwol W, Rimbert A, Wolters JC, Smit M, Bloks VW, Kloosterhuis NJ, Huijkman N, Koster MH, Tharehalli U, de Neck SM, Bournez C, Fuh MM, Kuipers J, Rajan S, de Bruin A, Ginsberg HN, van Westen GJP, Hussain MM, Scheja L, Heeren J, Zimmerman P, van de Sluis B, Kuivenhoven JA.
    Hepatology. 2022 Sep 2. [Pubmed]
    Van Zwol et al. identified small leucine-rich protein 1 (SLMR1) as a player in the VLDL biogenesis pathway and assessed its role by generating a liver-specific knockout mouse. Due to absence of available antibodies, the downregulation of Smlr1 was determined by targeted proteomics.
  • Absolute proteomic quantification reveals design principles of sperm flagellar chemosensation
    Trötschel C, Hamzeh H, Alvarez L, Pascal R, Lavryk F, Bönigk W, Körschen HG, Müller A, Poetsch A, Rennhack A, Gui L, Nicastro D, Strünker T, Seifert R, Kaupp UB.
    EMBO J. 2020 Feb 17;39(4) [PubMed]
    In this study, Trötschel et al. used a QconCAT from PolyQuant to absolutely quantify 19 proteins of a flagellar chemosensory signaling pathway from sperm of the sea urchin Arbacia punctulata.
  • QCAL--a novel standard for assessing instrument conditions for proteome analysis
    Eyers CE, Simpson DM, Wong SC, Beynon RJ, Gaskell SJ.
    J Am Soc Mass Spectrom. 2008 Sep;19(9):1275-80. [Pubmed]
    Original publication introducing the QCAL calibration standard for mass spectrometry instrumentations.
  • RePLiCal: A QconCAT Protein for Retention Time Standardization in Proteomics Studies
    Holman SW, McLean L, Eyers CE.
    J Proteome Res. 2016 Mar 4;15(3):1090-102 [Pubmed]
    Original publication introducing the RePLiCal retention time standard.
  • Platform Independent Protein-Based Cell-Of-Origin Subtyping of Diffuse Large B-cell Lymphoma in Formalin-Fixed Paraffin-Embedded Tissue
    Reinders J, Altenbuchinger M, Limm K, Schwarzfischer P, Scheidt T, Strasser L, Richter J, Szczepanowski M, Huber CG, Klapper W, Spang R, Oefner PJ.
    Sci Rep. 2020 May 12;10(1):7876. [Pubmed]
    In their study, Reinders et al. used RePLiCal for normalization of retention times by linear fitting.
  • Quantification of drug metabolising enzymes and transporter proteins in the paediatric duodenum via LC-MS/MS proteomics using a QconCAT technique
    Goelen J, Farrell G, McGeehan J, Titman CM, J W Rattray N, Johnson TN, Horniblow RD, Batchelor HK.
    Eur J Pharm Biopharm. 2023 Aug 23:S0939-6411(23)00214-X. [Pubmed]
    Goelen et al. used a QconCAT to simultaneously quantify 21 proteins of the three DMET-protein families (transporters, CYP and UGT-enzymes) using pinch biopsies from the paediatric duodenum.
  • Cov2MS: An Automated and Quantitative Matrix-Independent Assay for Mass Spectrometric Measurement of SARS-CoV-2 Nucleocapsid Protein
    Van Puyvelde B, Van Uytfanghe K, Van Oudenhove L, Gabriels R, Van Royen T, Matthys A, Razavi M, Yip R, Pearson T, Drouin N, Claereboudt J, Foley D, Wardle R, Wyndham K, Hankemeier T, Jones D, Saelens X, Martens G, Stove CP, Deforce D, Martens L, Vissers JPC, Anderson NL, Dhaenens M.
    Anal Chem. 2022 Dec 20;94(50):17379-17387. [Pubmed]
    Van Puyvelde et al. improved Cov-MS by adding SISCAPA technology to enrich proteotypic peptides of the SARS-CoV-2 nucleocapsid (N) protein from trypsin-digested patient samples. The Cov2MS assay is compatible with most matrices including nasopharyngeal swabs, saliva, and plasma, has increased sensitivity and a strong positive correlation with qPCR detection beyond a quantification cycle of 30-31.
  • A fast and sensitive absolute quantification assay for the detection of SARS-CoV-2 peptides using parallel reaction monitoring mass spectrometry
    Gajbhiye A, Nalbanta A, Heunisa T, Sidgwick F, Porter A, Tahab Y, Trost M
    Journal of Proteomics. Vol. 265, 15 August 2022. [Pubmed]
    Gajbhiyea et al. developed a high-throughput PRM-MS assay to directly detect viral peptides in nasopharyngeal swab samples. The assay enables sensitive detection and absolute quantification of SARS-CoV-2 nucleocapsid peptides with short turn-around times by using the Cov-MS isotopically labelled synthetic polypeptide as reference standard.
  • 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. 2022 Feb;88(4):1811-1823. [Pubmed]
    Vasilogianni et al. used QconCATs to quantify drug-metabolising enzymes, transporters, receptor tyrosine kinases (RTK) and protein markers in samples from colorectal cancer liver metastasis (CLRM) patients. The detected alterations in protein abundance may provide valuable information for diagnosis and therapeutic intervention.
  • Quantitative Proteomics of Hepatic Drug-Metabolizing Enzymes and Transporters in Patients with Colorectal Cancer Metastasis
    Vasilogianni AM, Al-Majdoub ZM, Achour B, Peters SA, Barber J, Rostami-Hodjegan A.
    Clin Pharmacol Ther. 2022 May 3. [Pubmed]
    Vasilogianni et al. studied the impact of liver cancer metastasis on protein abundance of 22 drug-metabolizing enzymes (DMEs) and 25 transporters. They used targeted proteomics and QconCATs as reference standards for analysis of microsome preparations from individuals and cancer patients.
  • A family of QconCATs (Quantification conCATemers) for the quantification of human pharmacological target proteins
    Vasilogianni AM, El-Khateeb E, Achour B, Alrubia S, Rostami-Hodjegan A, Barber J, Al-Majdoub ZM.
    J Proteomics. 2022 Jun 15;261:104572. [Pubmed]
    In this report, the authors describe the development and characterization of two QconCAT constructs for quantification of 24 enzymes and 21 receptor tyrosine kinases (RTKs), complementing two previously reported QconCATs for the quantification of key enzymes and drug transporters. The QconCATs were successfully applied in quantification of target proteins in human liver.
  • 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]
    Chatziioannou et al. used QconCATs and synthetic peptides belonging to 47 protein markers for a prospective case-control study evaluating serum proteomics profiles. They were able to define two panels of three proteins each that allow highly sensitive diagnosis of late-onset sepsis (LOS) and differential diagnosis between LOS and necrotizing enterocolitis.
  • 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]
    El-Katheeb et al. used QconCAT-based targeted proteomics to determine the absolute abundance of 51 drug-metabolizing enzymes and transporters in human liver microsomes across the three degrees for cirrhosis severity to determine their impact on the predictive performance of PBPK (physiologically based pharmacokinetic) models. Their work demonstrates the utility of proteomics-informed PBPK modeling for drug-specific dose adjustments in liver cirrhosis.
  • 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]
    The authors established a consortium consisting of 15 academic laboratories and several industrial partners to identify peptides for MS-based detection of SARS-CoV-2 infection. They describe the full pipeline for developing a fast and sensitive MS-based assay for detection of viral presence directly in clinical samples using conventional instrumentation.

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