Biomarker Discovery, Verification and Validation

In general, biomarker research follows a continuum that begins with discovery and proceeds through validation to the eventual implementation of biomarkers in a clinical setting. Biomarker discovery requires high confidence identification of biomarker candidates with simultaneous quantitation information to indicate which proteins are changing to a statistically relevant degree in response to disease. Biomarker candidates identified in discovery need to be validated using larger sample sets covering a broad section of patient cohorts. To avoid a potential bottleneck associated with taking a large number of candidates to validation, a verification step is employed to screen potential biomarkers to ensure that only the highest quality leads from the discovery phase are taken into the costly validation stage. The verification stage requires a high throughput workflow with a minimum of sample preparation that provides both high specificity and sensitivity. Additionally, the verification stage can confirm that a particular methodology is suitable to be used in the validation phase.

Biomarker Discovery Workflows

One of the biggest challenges in biomarker discovery is the difficulty of identifying medium to low abundance proteins in complex biological samples. For example, human plasma has over 1 X 106 different protein molecules with an estimated dynamic range approaching 1010. Of these, the 22 most abundant proteins make up 99% of the protein content of plasma, and the abundance of a given biomarker will fluctuate within a sample population. A robust biomarker discovery workflow must be capable of uncovering a panel of biomarkers in samples such as human plasma and cancer cell line lysates, with unambiguous identification and quantitative characterization. To address this challenge, quantitative proteomics workflows involving SILAC (Stable Isotope Labeling of Amino Acids in Culture) are employed. In the core facility, chiefly the Thermo Orbitrap Elite and Thermo LTQ Orbitrap Velos LC/MS systems are used for biomarker discovery.

Biomarker Verification Workflows

Because of normal clinical or biological variability, candidate biomarkers identified in the discovery stage need to be validated across a large number of samples. The challenge is to develop a fast, targeted analysis method capable of analyzing as many identified candidates as possible in minimally hundreds and potentially even thousands of samples. A biomarker candidate verification phase eliminates this bottleneck by ensuring that only the most promising putative biomarkers found in discovery go on to the validation stage. To address this challenge, targeted proteomics workflows involving MRM (Multiple Reaction Monitoring) and SISCAPA (Stable Isotope Standard with Capture by Anti-Peptide Antibodies) are employed on the Agilent 6490 iFunnel triple quadrupole LC/MS system.

Workflow Tutorials

Targeted LC/MS Methods

MALDI Applications

Mark Stolowitz, PhD
Director, Proteomics Core Facility
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