Reproducible Quantification of Cancer-Associated Proteins in Body Fluids Using Targeted Proteomics

Abstract

The rigorous testing of hypotheses on suitable sample cohorts is a major limitation in translational research. This is particularly the case for the validation of protein biomarkers where the lack of accurate, reproducible and sensitive assays for most proteins has precluded the systematic assessment of hundreds of potential marker proteins described in the literature. Here, we describe a high throughput method for the development and refinement of selected reaction monitoring (SRM) assays for human proteins. The method was applied to generate such assays for more than 1000 cancer-associated proteins, which are functionally related to candidate cancer driver mutations. We used the assays to determine the detectability of the target proteins in two clinically relevant samples, plasma and urine. 182 proteins were detected in depleted plasma, spanning five orders of magnitude in abundance and reaching below a concentration of 10 ng/mL. The narrower concentration range of proteins in urine allowed the detection of 408 proteins. Moreover, we demonstrate that these SRM assays allow the reproducible quantification of 34 biomarker candidates across 84 patient plasma samples. Through public access to the entire assay library, which will also be expandable in the future, researchers will be able to target their cancer-associated proteins of interest in any sample type using the detectability information in plasma and urine as a guide. The generated reference map of SRM assays for cancer-associated proteins is a valuable resource for accelerating and planning biomarker verification studies.

Proteomics of Microparticles with SILAC Quantification (PROMIS-Quan): A Novel Proteomic Method for Plasma Biomarker Quantification*

“…Among the identified proteins the microparticle proteome provided a rich source of potential biomarkers, as defined by Huttenhain et al (36). The intensities of these were reproducible and did not concentrate within the lower range of intensities, but rather showed a wide range (Fig.…”

Quantitative Mass Spectrometric Profiling of Cancer-cell Proteomes Derived From Liquid and Solid Tumors

“…If the required acquisition time required could be reduced from 24 -36 hr (liquid tumors) or 3 hr (solid tumors) to e.g., 1 hr using targeted mass spectrometry and a simple one-dimensional separation of peptides, then the resulting gain in throughput could be used to increase significantly the numbers of biological and technical replicates examined, with corresponding improvements in the significance of quantification results. Targeted approaches have already been demonstrated to be a suitable tool for the verification of cancer-associated protein biomarker-candidates 22 , and have been developed to a point where they show promise for validation or even as a potential tool for routine clinical use 23,24 .…”

Personalisierte Medizin

“…Vi rientrano anche l'importanza futura dei diritti della personalità, le questioni di equità correlate allo sviluppo della Medicina Personalizzata nonché la comprensione della salute e della malattia. (Topol, 2012 (Huttenhain et al, 2012 Systembiologie ist eine verhältnismässig junge Disziplin, die sich seit Ende der 1990er Jahre kontinuierlich weiterentwickelt hat (Döring, 2012, S. 36 (Franke, 2012).…”