The Path to Clinical Proteomics Research: Integration of Proteomics, Genomics, Clinical Laboratory and Regulatory Science

Better biomarkers are urgently needed to cancer detection, diagnosis, and prognosis. While the genomics community is making significant advances in understanding the molecular basis of disease, proteomics will delineate the functional units of a cell, proteins and their intricate interaction network and signaling pathways for the underlying disease. Great progress has been made to characterize thousands of proteins qualitatively and quantitatively in complex biological systems by utilizing multi-dimensional sample fractionation strategies, mass spectrometry and protein microarrays. Comparative/quantitative analysis of high-quality clinical biospecimen (e.g., tissue and biofluids) of human cancer proteome landscape has the potential to reveal protein/peptide biomarkers responsible for this disease by means of their altered levels of expression, post-translational modifications as well as different forms of protein variants. Despite technological advances in proteomics, major hurdles still exist in every step of the biomarker development pipeline. The National Cancer Institute's Clinical Proteomic Technologies for Cancer initiative (NCI-CPTC) has taken a critical step to close the gap between biomarker discovery and qualification by introducing a pre-clinical "verification" stage in the pipeline, partnering with clinical laboratory organizations to develop and implement common standards, and developing regulatory science documents with the US Food and Drug Administration to educate the proteomics community on analytical evaluation requirements for multiplex assays in order to ensure the safety and effectiveness of these tests for their intended use

Testing Suitability of Cell Cultures for SILAC-Experiments Using SWATH-Mass Spectrometry

Precise quantification is a major issue in contemporary proteomics. Both stable-isotope-labeling and label-free methods have been established for differential protein quantification and both approaches have different advantages and disadvantages. The present protocol uses the superior precision of label-free SWATH-mass spectrometry to test for suitability of cell lines for a SILAC-labeling approach as systematic regulations may be introduced upon incorporation of the "heavy" amino acids. The SILAC-labeled cell cultures can afterwards be used for further analyses where stable-isotope-labeling is mandatory or has substantial advantages over label-free approaches such as pulse-chase-experiments and differential protein interaction analyses based on co-immunoprecipitation. As SWATH-mass spectrometry avoids the missing-value-problem typically caused by undersampling in highly complex samples and shows superior precision for the quantification, it is better suited for the detection of systematic changes caused by the SILAC-labeling and thus, can serve as a useful tool to test cell lines for changes upon SILAC-labeling

Systematic quantification of peptides/proteins in urine using selected reaction monitoring

International audienceThe 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