QconCAT: Internal Standard for Protein Quantification.

Protein quantification based on stable isotope labeling-mass spectrometry involves adding known quantities of stable isotope-labeled internal standards into biological samples. The internal standards are analogous to analyte molecules and quantification is achieved by comparing signals from isotope-labeled and analyte molecules. This methodology is broadly applicable to proteomics research, biomarker discovery and validation, and clinical studies, which require accurate and precise protein abundance measurements. One such internal standard platform for protein quantification is concatenated peptides (QconCAT). This chapter describes a protocol for the design, expression, characterization, and application of the QconCAT strategy for protein quantification.

[1]  R. Beynon,et al.  Multiplexed absolute quantification for proteomics using concatenated signature peptides encoded by QconCAT genes , 2006, Nature Protocols.

[2]  Craig Lawless,et al.  Global absolute quantification of a proteome: Challenges in the deployment of a QconCAT strategy , 2011, Proteomics.

[3]  F. Vandenesch,et al.  Isotope-labeled Protein Standards , 2007, Molecular & Cellular Proteomics.

[4]  R. Aebersold,et al.  Quantotypic properties of QconCAT peptides targeting bovine host response to Streptococcus uberis. , 2012, Journal of Proteome Research.

[5]  R. Beynon,et al.  Multiplexed absolute quantification in proteomics using artificial QCAT proteins of concatenated signature peptides , 2005, Nature Methods.

[6]  I. Turko,et al.  Natural flanking sequences for peptides included in a quantification concatamer internal standard. , 2015, Analytical chemistry.

[7]  Hannah Johnson,et al.  Rigorous determination of the stoichiometry of protein phosphorylation using mass spectrometry , 2009, Journal of the American Society for Mass Spectrometry.

[8]  Henry H. N. Lam,et al.  PeptideAtlas: a resource for target selection for emerging targeted proteomics workflows , 2008, EMBO reports.

[9]  C. Eyers,et al.  Quantification of proteins and their modifications using QconCAT technology. , 2011, Methods in enzymology.

[10]  Sriram Subramaniam,et al.  Stoichiometry and Absolute Quantification of Proteins with Mass Spectrometry Using Fluorescent and Isotope-labeled Concatenated Peptide Standards*S , 2008, Molecular & Cellular Proteomics.

[11]  S. Gaskell,et al.  Quantification of the proteins of the bacterial ribosome using QconCAT technology. , 2014, Journal of proteome research.

[12]  Ruedi Aebersold,et al.  Comparative Evaluation of Current Peptide Production Platforms Used in Absolute Quantification in Proteomics*S , 2008, Molecular & Cellular Proteomics.

[13]  S. Rutherfurd,et al.  Amino Acid Analysis , 2009, Current protocols in protein science.

[14]  Johanna E. Camara,et al.  Expression and characterization of 15N-labeled human C-reactive protein in Escherichia coli and Pichia pastoris for use in isotope-dilution mass spectrometry. , 2012, Protein expression and purification.

[15]  I. Turko,et al.  Mass spectrometry quantification of clusterin in the human brain , 2012, Molecular Neurodegeneration.

[16]  I. Turko,et al.  Trends in QconCATs for targeted proteomics , 2014 .

[17]  E. Mandelkow,et al.  Quantification of Amyloid Precursor Protein and Tau for the Study of Axonal Traffic Pathways , 2007, The Journal of Neuroscience.

[18]  I. Turko,et al.  Quantification of amyloid precursor protein isoforms using quantification concatamer internal standard. , 2013, Analytical chemistry.

[19]  RockOn Team,et al.  Re: Attenuation compensation in single-photon emission tomography: a comparative evaluation. , 1983, Journal of nuclear medicine : official publication, Society of Nuclear Medicine.

[20]  Robert J Beynon,et al.  QconCATs: design and expression of concatenated protein standards for multiplexed protein quantification , 2012, Analytical and Bioanalytical Chemistry.

[21]  I. Turko,et al.  Quantitative performance of internal standard platforms for absolute protein quantification using multiple reaction monitoring-mass spectrometry. , 2015, Analytical chemistry.

[22]  R D Appel,et al.  Protein identification and analysis tools in the ExPASy server. , 1999, Methods in molecular biology.

[23]  L. Martens,et al.  Getting intimate with trypsin, the leading protease in proteomics. , 2013, Mass spectrometry reviews.

[24]  Philip Brownridge,et al.  The importance of the digest: proteolysis and absolute quantification in proteomics. , 2011, Methods.