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Clinical proteomics

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Vincent Delatour
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Clinical mass spectrometry proteomics (cMSP) assays are being increasingly used in clinical laboratories for analyzing peptides and proteins. It has therefore become urgent to characterize and validate the methods available for liquid chromatography–tandem mass spectrometry (LC/MS–MS) targeted quantification of peptide and protein biomarkers in biological fluids in the context of in vitro diagnostics. LC–MS/MS for the detection of peptides and proteins is currently the main approach used in the field of cMSP. As a result of their selectivity, low reagent costs and the fact that these methods can be used for absolute quantification and multiplexing, they will likely eventually replace immunoassays. Although LC–MS/MS is known to be the main reference method involved in reference measurement procedures (RMPs), it needs to meet the requirements of in vitro diagnostic (IVD) regulations and standards. This review shows that cMSP is fully compatible with the regulatory IVD requirements and provides an overview of the characterization and validation of the use of LC–MS/MS targeted quantification of clinical protein biomarkers in biological fluids.
Jerome Vialaret
added a research item
Background: Monoclonal antibody (mAb) is the first class of biotechnological products followed by recombinant proteins and vaccines. Bevacizumab (AVASTIN) product by Roche, is able to neutralize Vascular Endothelial Growth factor (VEGF), a regulator of angiogenesis and surexpressed in a lot of human tumors. By combining a step of Protein-A capture and a LC-MRM analysis, Bevacizumab can be quantified on patient serum during cure (cycle 1, 3, 5, 7, 9 and 11). Methods: The analytical strategy was designed to quantify the total fraction of bevacizumab kinetics in human serum. Sample pretreatment was necessary for the quantitation of mAb due to the complexity of human plasma matrix. Two approaches were developed: (i) an automated Prot-A purification workflow (Bravo Assay map Agilent) followed by a trypsin digest and LC-MRM analysis; and (ii) the use of a commercially available kit based on nano-surface and molecular-orientation limited (nSMOL) proteolysis. Both approach have been validated and compared. 10 min LC-MRM runs were performed on a 8060 TQ from Shimadzu. Data were normalized using SILuTMMAb as internal standard and compared with 23 patients’ serum dosed using ELISA assay. Results: Unique proteotypic peptide (FTFSLDTSK) was selected for the two approaches and for the quantification in agreement with previously published work (1; 2). Both methods were validated on linear range 1.957 to 766.667 µg/ml for the automated Prot-A purification workflow, and 0.269 to 766.667 µg/ml for the nSMOL workflow. The two workflows were analytically validated in agreement with international guidelines. nSMOL workflow gave a 10-fold lower LLOQ, a more linear calibration curve but is a little bit less accurate (96.33% against 100.59%) than the automated Prot-A purification workflow. The use of nSMOL workflow was finally less time consuming and easier to perform. The 2 workflows were compatible with high throughput LC-MRM analyses of mAbs. Conclusions: The two workflows exhibited similar quantitative performance considering the low number of patient samples analyzed. Both methods used a limited sample volume (5µL) and are suitable for clinical application.
Jerome Vialaret
added a research item
Monoclonal antibody (mAb) is the first class of biotechnological products followed by recombinant proteins and vaccines. Bevacizumab (AVASTIN®) product by Roche, is able to neutralize Vascular Endothelial Growth factor (VEGF), a regulator of angiogenesis and surexpressed in a lot of human tumors. The analytical strategy was designed to quantify the total fraction of bevacizumab kinetics in human serum using a sample preparation protocol to prefractionate the serum sample. An in-house protocol developed using AssayMAP Bravo platform (automated protein-A purification and Trypsin/LysC mixed digestion; Agilent Technologies) was compared to a ready-to-use commercial kit (nano-surface and molecular-orientation limited (nSMOL®) proteolysis, which is a Fab-selective limited proteolysis approach; Shimadzu Corporation). Bevacizumab can be accurately quantified with these two technologies on patient serum during cure (cycle 1, 3, 5, 7, 9, and 11).
Jerome Vialaret
added a research item
Analytical performances comparison of two different mAbs preparation for clinical mass spectrometry quantification: Fully Automatized immune-enrichment based on Protein-A Tips and commercially available nSMOL preparation (Shimadzu)
Sylvain Lehmann
added 5 research items
This manuscript reports the conclusions of a working group of the French Society of Clinical Biology (SFBC) 2007-2008 which evaluated the status, the impact and the future developments related to the analysis of the proteome using multiplexed methods. These approaches that are also used in clinical biology are performed on solid supports or in flow by using specifically dedicated or conventional flow cytometers. This review provides therefore a broad overview going from the methods already present in research laboratories to the tools for clinical and medical investigations.
Research of new diagnosis or prognosis biomarkers is a major challenge for the management of patients with complex pathologies like cancer. Clinical proteomics is one of the recent approaches to identify these biomarkers in biological fluids. Over the last five years, many problems related to the variability and the quality control of these analyses have been observed. This was notably related to the different preanalytical status of each sample. A strong need for standardization of the critical preanalytical phases (collection, transport, processing, storage...) has been therefore recognized. With this goal in mind, working groups of the "Institut national du cancer" (INCa) and the "Société française de biologie clinique" (SFBC) proposed here preanalytical proteomics guidelines for the most common biological fluids: plasma, serum, urine and cerebrospinal fluid. To goal is to provide the basis for the harmonization of the procedures in clinical laboratories and biobanks to allow an optimal use of biological collections.
Clinical Proteomics biomarker discovery programs lead to the selection of putative new biomarkers of human pathologies. Following an initial discovery phase, validation of these candidates in larger populations is a major task that recently started relying upon the use of mass spectrometry approaches, especially in cases where classical immune-detection methods were lacking. Thanks to highly sensitive spectrometers, adapted measurement methods like selective reaction monitoring (SRM) and various pre-fractionation methods, the quantitative detection of protein/peptide biomarkers in low concentrations is now feasible from complex biological fluids. This possibility leads to the use of similar methodologies in clinical biology laboratories, within a new proteomic field that we shall name "Clinical Chemistry Proteomics" (CCP). Such evolution of Clinical Proteomics adds important constraints with regards to the in vitro diagnostic (IVD) application. As measured values of analytes will be used to diagnose, follow-up and adapt patient treatment on a routine basis; medical utility, robustness, reference materials and clinical feasibility are among the new issues of CCP to consider.
Sylvain Lehmann
added 2 research items
Abstract Proteomics studies typically aim to exhaustively detect peptides/proteins in a given biological sample. Over the past decade, the number of publications using pro-teomics methodologies has exploded. This was made possible due to the availability of high-quality genomic data and many technological advances in the fields of microfluidics and mass spectrometry. Proteomics in biomedical research was initially used in 'functional' studies for the identification of proteins involved in pathophysiological processes, complexes and networks. Improved sensitivity of instrumentation facilitated the analysis of even more complex sample types, including human biological fluids. It is at that point the field of clinical proteomics was born, and its fundamental aim was the discovery and (ideally) validation of biomarkers for the diagnosis, prognosis, or therapeutic monitoring of disease. Eventually, it was recognized that the technologies used in clinical proteomics studies [particularly liquid chromatography-tandem mass spectrometry (LC-MS/MS)] could represent an alternative to classical immunochemical assays. Prior to deploying MS in the measurement of peptides/proteins in the clinical laboratory, it seems likely that traditional proteo-mics workflows and data management systems will need to adapt to the clinical environment and meet in vitro diagnostic (IVD) regulatory constraints. This defines a new field, as reviewed in this article, that we have termed quantitative Clinical Chemistry Proteomics (qCCP).