Researchers seeking to exploit the plasma proteome for diagnostic, drug discovery, and related applications face analytical challenges arising out of the wide concentration range and structural complexity of its constituent proteins, as well as the limitations of current analytical techniques. The concentration range of proteins in human plasma spans approximately twelve orders of magnitude, with 85 to 90% of the protein mass distributed across as few as six proteins. Specialized affinity columns have simplified the removal of high-abundance proteins, facilitating an investigation of the large number of low-abundance proteins present in the immunodepleted sample.1
Immunodepeleted plasma represents a complex mixture of hundreds or thousands of proteins. Fractionation of the sample is an important step in reducing the complexity pursuant to analysis. Given the low abundance of the analytes of interest (pg/mL to ng/mL range), any fractionation technique must provide high recoveries, efficient separations, and reproducibility, especially if the objective is the validation of protein biomarkers — a task considered key to understanding cellular and tissue dysfunction and exploring consequent pathogenicity. Current separation methods such as two-dimensional gel electrophoresis (2DGE) and two-dimensional liquid chromatography (2DLC) are limited in these applications by inefficient sample recoveries and poor reproducibility. The poor reproducibility associated with 2DGE and reverse phase (RP) chromatography often makes it difficult to directly compare the protein content of samples.