Dataset Information

Comprehensive human urine standards for comparability and standardization in clinical proteome analysis.

ABSTRACT: Urine proteomics is emerging as a powerful tool for biomarker discovery. The purpose of this study is the development of a well-characterized "real life" sample that can be used as reference standard in urine clinical proteomics studies.We report on the generation of male and female urine samples that are extensively characterized by different platforms and methods (CE-MS, LC-MS, LC-MS/MS, 1-D gel analysis in combination with nano-LC MS/MS (using LTQ-FT ultra), and 2-DE-MS) for their proteome and peptidome. In several cases analysis involved a definition of the actual biochemical entities, i.e. proteins/peptides associated with molecular mass and detected PTMs and the relative abundance of these compounds.The combination of different technologies allowed coverage of a wide mass range revealing the advantages and complementarities of the different technologies. Application of these samples in "inter-laboratory" and "inter-platform" data comparison is also demonstrated.These well-characterized urine samples are freely available upon request to enable data comparison especially in the context of biomarker discovery and validation studies. It is also expected that they will provide the basis for the comprehensive characterization of the urinary proteome.

SUBMITTER: Mischak H

PROVIDER: S-EPMC3064949 | biostudies-literature | 2010 Apr

REPOSITORIES: biostudies-literature

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Comprehensive human urine standards for comparability and standardization in clinical proteome analysis.

Mischak Harald H Kolch Walter W Aivaliotis Michalis M Bouyssié David D Court Magali M Dihazi Hassan H Dihazi Gry H GH Franke Julia J Garin Jérôme J Gonzalez de Peredo Anne A Iphöfer Alexander A Jänsch Lothar L Lacroix Chrystelle C Makridakis Manousos M Masselon Christophe C Metzger Jochen J Monsarrat Bernard B Mrug Michal M Norling Martin M Novak Jan J Pich Andreas A Pitt Andrew A Bongcam-Rudloff Erik E Siwy Justyna J Suzuki Hitoshi H Thongboonkerd Visith V Wang Li-Shun LS Zoidakis Jérôme J Zürbig Petra P Schanstra Joost P JP Vlahou Antonia A

Proteomics. Clinical applications 20100203 4

<h4>Purpose</h4>Urine proteomics is emerging as a powerful tool for biomarker discovery. The purpose of this study is the development of a well-characterized "real life" sample that can be used as reference standard in urine clinical proteomics studies.<h4>Experimental design</h4>We report on the generation of male and female urine samples that are extensively characterized by different platforms and methods (CE-MS, LC-MS, LC-MS/MS, 1-D gel analysis in combination with nano-LC MS/MS (using LTQ-F ...[more]

PMID: 21137064

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Project description:Comparability studies lie at the heart of assessments that evaluate differences amongst manufacturing processes and stability studies of protein therapeutics. Low resolution chromatographic and electrophoretic methods facilitate quantitation, but do not always yield detailed insight into the effect of the manufacturing change or environmental stress. Conversely, mass spectrometry (MS) can provide high resolution information on the molecule, but conventional methods are not very quantitative. This gap can be reconciled by use of a stable isotope-tagged reference standard (SITRS), a version of the analyte protein that is uniformly labeled (13)C6-arginine and (13)C6-lysine. The SITRS serves as an internal control that is trypsin-digested and analyzed by liquid chromatography (LC)-MS with the analyte sample. The ratio of the ion intensities of each unlabeled and labeled peptide pair is then compared to that of other sample(s). A comparison of these ratios provides a readily accessible way to spot even minute differences among samples. In a study of a monoclonal antibody (mAb) spiked with varying amounts of the same antibody bearing point mutations, peptides containing the mutations were readily identified and quantified at concentrations as low as 2% relative to unmodified peptides. The method is robust, reproducible and produced a linear response for every peptide that was monitored. The method was also successfully used to distinguish between two batches of a mAb that were produced in two different cell lines while two batches produced from the same cell line were found to be highly comparable. Finally, the use of the SITRS method in the comparison of two stressed mAb samples enabled the identification of sites susceptible to deamidation and oxidation, as well as their quantitation. The experimental results indicate that use of a SITRS in a peptide mapping experiment with MS detection enables sensitive and quantitative comparability studies of proteins at high resolution.

Dataset Information

Comprehensive human urine standards for comparability and standardization in clinical proteome analysis.

Publications

Comprehensive human urine standards for comparability and standardization in clinical proteome analysis.

Similar Datasets

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