title: Protein quantification using a cleavable reporter peptide.
abstract: Peptide and protein quantification based on isotope dilution and mass spectrometry analysis are widely employed for the measurement of biomarkers and in system biology applications. The accuracy and reliability of such quantitative assays depend on the quality of the stable-isotope labeled standards. Although the quantification using stable-isotope labeled peptides is precise, the accuracy of the results can be severely biased by the purity of the internal standards, their stability and formulation, and the determination of their concentration. Here we describe a rapid and cost-efficient method to recalibrate stable isotope labeled peptides in a single LC-MS analysis. The method is based on the equimolar release of a protein reference peptide (used as surrogate for the protein of interest) and a universal reporter peptide during the trypsinization of a concatenated polypeptide standard. The quality and accuracy of data generated with such concatenated polypeptide standards are highlighted by the quantification of two clinically important proteins in urine samples and compared with results obtained with conventional stable isotope labeled reference peptides. Furthermore, the application of the UCRP standards in complex samples is described.
authors.affiliation: Luxembourg Clinical Proteomics Center, CRP-Santé , 1 A-B rue Thomas Edison, Strassen 1445, Luxembourg.
authors.forename: Elodie
authors.initials: E
authors.lastname: Duriez
authors.forename: Stephane
authors.initials: S
authors.lastname: Trevisiol
authors.forename: Bruno
authors.initials: B
authors.lastname: Domon
mesh_terms: D000328:Adult; D000595:Amino Acid Sequence; D002138:Calibration; D002853:Chromatography, Liquid; D005260:Female; D006801:Humans; D007553:Isotope Labeling; D008297:Male; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010455:Peptides / Q000032:analysis* / Q000737:chemistry; D011506:Proteins / Q000032:analysis* / Q000737:chemistry; D012015:Reference Standards
chemical_list: D010455:Peptides; D011506:Proteins
keywords: calibration; cleavable reporter peptide; mass spectrometry; peptide/protein quantification; stable isotope labeled peptides; standard recalibration; targeted proteomics