Translational proteomics has not been as successful as originally anticipated. Because mass spectrometry (MS) can separate
proteins at the sequence level, it provides the selectivity needed for this application; however, traditional challenges still
exist, including time-to-result, throughput, and sample-size requirements. For analytical validation and verification purposes,
sample preparation times must be reduced from days to hours. Scientists recently coupled a previously developed immunoaffinity
enrichment method to selected reaction monitoring (SRM) MS. The MS immunoassay–SRM method combines liquid chromatography–tandem
mass spectrometry (LC–MS-MS) for target identification, a microscale immunoaffinity capture method for enrichment, and subsequent
SRM analysis. Using the MS immunoassay–SRM workflow, a standard high-throughput method for developing targeted biomarker identification
of proteins in human plasma and serum for clinical research was developed.
Mass spectrometry (MS)-driven proteomics has made progress in the identification and quantification of disease biomarkers,
including C-reactive protein as an indicator for myocardial infarction and prostate-specific antigen (PSA) for prostate cancer.
Despite these and other successes, translational proteomics, which is defined as the translation of biomarker discovery to routine analysis, has not been nearly as successful as originally
anticipated because comprehensive proteomic analysis of plasma, serum, and other biological fluids has proved exceedingly
challenging. The "look alike" nature of molecular isoforms, the enormous dynamic range of protein concentrations of potential
interest (>10 orders of magnitude in blood plasma), and the fact that molecules of interest are often in low abundance have
all slowed the progress of biomarker hunters across the globe.
One example of the challenge presented by protein analyte isoforms is demonstrated by the need to distinguish between full-length
parathyroid hormone (PTH) 1-84 and multiple N-terminally truncated PTH variants. The differences between these isoforms are
critical to accurate diagnosis of endocrine and osteological diseases (1). Similarly, in clinical testing, PSA typically presents
in truncated and modified isoforms, making precise detection and quantification difficult which contributes to a high false-positive
From a processing point of view, binding protein detachment and the high dynamic range of samples have hampered assays
for insulin-like growth factor 1 (IGF-1), a marker for growth-related illness that is important in cell proliferation, differentiation,
apoptosis, and tissue growth from a research point of view (3).
To add to these challenges, verification and population-scale biomarker validation require the analysis of hundreds or even
thousands of high-quality samples. Sample collection and storage must use standard protocols to reduce potential variations
attributable to endogenous enzymes or sample contamination. Verification and validation studies require multiple control groups
and subjects in disease subcategories — all gathered over the course of disease progression. The analysis of many samples
is required to distinguish normal human genetic heterogeneity and heterogeneity attributable to disease. High-throughput detection
methods are essential to achieving statistical confidence in the accurate identification of molecules of interest (4).