Enhanced Affinity Columns Simplify Protein Fractionation

Columns enable reproducible chromatographic separation with high recovery of low-abundance plasma proteins.
Aug 01, 2005
Volume 18, Issue 8

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.

Table 1. Experimental Details
MACROPOROUS REVERSED-PHASE PROTEIN FRACTIONATION Limitations of current protein fractionation methods are driving the development of more effective separation techniques, especially for resolving complex mixtures of low-abundance proteins. A new approach to this problem utilizes a recently developed macroporous reversed-phase chromatographic column (mRP-C18, Agilent Technologies). The column fractionates plasma samples previously immunodepleted of their high-abundance proteins, with high protein recovery, good resolution, and excellent reproducibility. The quality of the separation enables rapid prescreening and differentiation of immunodepleted samples by examining their chromatographic UV profiles. In this study, three immunodepleted sera — a control from a healthy subject, a cortisol-deficient serum (Sigma No. 7269), and a high rheumatoid factor serum (Sigma No. 3145) — were fractionated and their UV profiles were compared for differences. Fractions with major differences from each sample were analyzed by LC/MS while the remaining samples were stored for further analysis. An independent assessment of protein recoveries also was performed. Table 1 lists equipment and materials employed in the separations and details the experimental protocol. Except where noted, our company supplied equipment and materials.

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