Table 2. Recovery of Serum Proteins as Determined by the BCA Assay
Figure 1. Fractionation Recovery. Two chromatograms show the separation of immunodepleted serum (blue) followed by a blank
injection (red). The high recovery from the mRP-C18 column is demonstrated by the absence of significant absorbance in the
RESULTS AND DISCUSSION
When dealing with the fractionation of complex mixtures of low-abundance proteins, there is an important interplay between
resolution and recovery. mRP-C18 protein fractionation with high recovery enables a significant reduction in sample complexity
prior to analysis without loss of important components. This, in turn, enhances the effectiveness of the post-digestion liquid
chromatography-mass spectrometry (LC-MS) analysis. High recovery from a column is also critical in preventing cross-contamination
of subsequent samples due to carryover. This can be problematic in protein separations by traditional RP high-performance
(HP) LC methods, with typical recoveries ranging from 30 to 80%. High recovery is also important in the validation of biomarkers,
allowing for direct quantification of concentration differences between control and treated or diseased samples. Data for
samples processed with a mRP-C18 column show 99 percent recovery (Table 2). Analysis of chromatograms from a sample followed by a blank sample showed no significant carryover, demonstrating greater
than 99% recovery of serum proteins (Figure 1). The results provide confidence that this new technique can be used to compare
diseased and control samples free of carryover contamination.
Figure 2. Fractionation Reproducibility. Overlay of mRP-C18 column chromatograms for five consecutive injections. Inset shows
chromatogram overlay for two injections of the same serum sample performed one week apart. Column was used extensively in
the intervening period.
Good reproducibility is essential if a protein fractionation technique is to be used to detect, validate, or compare potential
protein biomarkers in serum. To evaluate mRP-C18 reproducibility, the column was subjected to five consecutive injections
of an immunodepleted serum sample and chromatographed under optimized conditions for protein fractionation. The results are
shown in Figure 2. The figure insert verifies a robust reproducibility of the column by comparing the results of two samples
of the same immunodepleted serum injected one week apart. This test was made even more rigorous by allowing the column to
be used extensively for unrelated experiments during the intervening period.
Figure 3. Biomarker Detection and Screening. Immunodepleted serum samples of control serum (blue) cortisol-deficient (red)
and high rheumatoid factor serum (green) as fractionated on the mRP-C18 column. Inset shows the expanded range of chromatograms
with obvious differences in UV absorbance. Protein loads for each sample were 300 mg.
A control serum sample, a cortisol-deficient serum sample, and an elevated rheumatoid factor serum, all identically immunodepleted
of high-abundance proteins, were injected and separated on an mRP-C18 column. Chromatograms were overlaid and analyzed for
differences in their UV absorbancy profiles. The high-resolution fractionation the column produces demonstrates its utility
as a rapid screening tool for identifying and comparing sample fractions by means of UV chromatographic differences. Remaining
fractions are stored for further analysis (Figure 3).