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.
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.
Table 1. Experimental Details