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Mass spectrometry–based biomarker discovery in bio-fluids produces a list of candidate proteins that must be verified and quantified in a large number of samples before a candidate becomes a useful diagnostic, prognostic, or pharmacodynamic marker. Because of the high sensitivity and specificity provided by multiple reaction monitoring (MRM), this MS/MS method has recently been used for verification and quantification of potential biomakers. However, the wide dynamic range of protein concentrations in serum prohibits direct detection of many useful biomarkers at the concentration level of low nanogram/mL to picogram/mL range without any sample fractionation and/or enrichment.
In this presentation, we evaluate the utility of two sample enrichment techniques for improving the limit of detection and limit of quantification (LOQ) for MRM analysis of several candidate protein biomarkers.
In the first sample enrichment method, we used immuno-depletion to remove either the six most abundant serum proteins (90% serum depletion) or the twenty most abundant proteins (97% serum depletion) before MRM analysis of low-abundance potential biomarkers. The second sample enrichment method that we evaluated was the glycoprotein affinity enrichment method. Several low-abundance serum proteins were quantified by the MRM method using a triple quadrupole mass spectrometer coupled to a nanoscale liquid chromatograph. The effects of immuno-depletion and affinity enrichment on the LOQ of selected candidate proteins biomarkers in human plasma were compared.