title: Systematic quantification of peptides/proteins in urine using selected reaction monitoring.
abstract: The evaluation of biomarkers in bodily fluids necessitates the development of robust methods to quantify proteins in a complex background, using large sets of samples. The ability to multiplex numerous analytes in a single assay expedites the process. Liquid chromatography-mass spectrometry (LC-MS) analyses performed in selected reaction monitoring (SRM) in conjunction with stable isotope dilution MS present an effective way to detect and quantify biomarker candidates in bodily fluids. The strategy presented involves an initial qualification of predefined sets of proteins in urine. The technique was applied to detect and quantify peptides in urine samples as surrogates for a few endogenous proteins. Multiplexed assays were developed to analyze proteins associated with bladder cancer; a few exogenous proteins were added as internal standards. The sample preparation and the analytical protocols were optimized to ensure reproducibility, analytical precision, and quantification limits in the low nanogram per milliliter range. Analyses were performed using known amounts of isotopically labeled peptides. Systematic replication of the measurements indicated intra-assay and inter-assay variability, with CVs in the range of 10%. The differences measured for two targeted proteins were correlated with their level of expression in the corresponding tumors using immunohistochemistry.
authors.affiliation: Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland.
authors.forename: Nathalie
authors.initials: N
authors.lastname: Selevsek
authors.forename: Mariette
authors.initials: M
authors.lastname: Matondo
authors.forename: Marta
authors.initials: M
authors.lastname: Sanchez Carbayo
authors.forename: Ruedi
authors.initials: R
authors.lastname: Aebersold
authors.forename: Bruno
authors.initials: B
authors.lastname: Domon
mesh_terms: D000595:Amino Acid Sequence; D000704:Analysis of Variance; D014408:Biomarkers, Tumor / Q000652:urine; D016022:Case-Control Studies; D002853:Chromatography, Liquid; D051152:Clusterin / Q000652:urine; D003627:Data Interpretation, Statistical; D006801:Humans; D018960:Hyaluronan Receptors / Q000652:urine; D007150:Immunohistochemistry; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000592:standards / Q000652:urine*; D011507:Proteinuria / Q000652:urine; D040901:Proteomics / Q000379:methods / Q000592:standards* / Q000706:statistics & numerical data; D011786:Quality Control; D012015:Reference Standards; D015203:Reproducibility of Results; D053719:Tandem Mass Spectrometry / Q000592:standards / Q000706:statistics & numerical data; D001749:Urinary Bladder Neoplasms / Q000652:urine; D014556:Urine / Q000737:chemistry
chemical_list: D014408:Biomarkers, Tumor; C497483:CD44 protein, human; C495242:CLU protein, human; D051152:Clusterin; D018960:Hyaluronan Receptors; D010455:Peptides