The identification of novel biomarkers from human plasma remains a critical\nneed in order to develop and monitor drug therapies for nearly all disease areas. The\ndiscovery of novel plasma biomarkers is, however, significantly hampered by the\ncomplexity and dynamic range of proteins within plasma, as well as the inherent variability\nin composition from patient to patient. In addition, it is widely accepted that most soluble\nplasma biomarkers for diseases such as cancer will be represented by tissue leakage\nproducts, circulating in plasma at low levels. It is therefore necessary to find approaches\nwith the prerequisite level of sensitivity in such a complex biological matrix. Strategies for\nfractionating the plasma proteome have been suggested, but improvements in sensitivity\nare often negated by the resultant process variability. Here we describe an approach using\nmultidimensional chromatography and on-line protein derivatization, which allows for\nhigher sensitivity, whilst minimizing the process variability. In order to evaluate this\nautomated process fully, we demonstrate three levels of processing and compare\nsensitivity, throughput and reproducibility. We demonstrate that high sensitivity analysis of\nthe human plasma proteome is possible down to the low ng/mL or even high pg/mL level\nwith a high degree of technical reproducibility.
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