As molecular biomarkers go, proteins are possibly the most universally affected by disease state.  Protein signatures encompass not only total levels of expression, but predominance of different isoforms, cleavage products, or post-translational modifications (e.g., phosphorylation, methylation, glycosylation, ubiquitination, etc.).  The dynamic sensitivity of these molecular profiles to the health status of the patient creates a bewilderingly complex web of information from which to select the most useful biomarkers.

Researchers have relied heavily on proteomic methods, including mass spectrometry and affinity-based technologies (i.e., multiplex immunoassays) in the hunt for biomarker candidates to feed into the pipeline of discovery.  Mass spectrometry techniques, particularly variants of LC-MS, seek to identify proteins on the back end – that is to say, every peptide fragment that is successfully ionized is identified by its inherent charge-to-mass ratio.  Multiplex immunoassays on the other hand, seek candidate proteins on the front end, by using a pre-selected panel of antibodies to target markers that have known or suspected relevance in disease processes.

Each of the 2 methods has different attributes in terms of sensitivity, throughput, and breadth of content. While LC-MS offers the possibility of truly de novo discoveries of key proteins, multiplex immunoassay enables reproducible detection of low-abundance analytes such as cytokines and other secreted effectors.  Mass spectrometry and antibody arrays may thus target different subsets of the proteome and for this reason, are sometimes used in tandem to broaden the screen1-3.

Despite persistent efforts by researchers, identifying clinically significant protein biomarkers has proven a highly challenging task. One major roadblock arises in the validation phase: using LC-MS methods, it can be difficult to follow up on positive hits if no reliable probes are available (see infographic below). However, one important advancement may serve to facilitate the process: an increase in the number of rigorously tested, commercially available antibodies. This not only expands the selection of immunoassays for the validation phase but can broaden the initial biomarker screen by enabling ever larger array panels. And importantly, the use of antibody arrays for the discovery phase facilitates downstream verification and validation experiments by providing a reliable antibody for every target. Currently, panels are available with >1000 sandwich antibody pairs or >2000 single antibodies, and those numbers continue to increase. These high-density arrays are primarily being developed by commercial suppliers in the hopes of accelerating the pipeline of current and future clinically viable biomarkers.

Infographic (click it to view full screen):




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