Enhanced Affinity Columns Simplify Protein Fractionation

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BioPharm International, BioPharm International-08-01-2005, Volume 18, Issue 8

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.

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.


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


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).

Table 2. Recovery of Serum Proteins as Determined by the BCA AssayAnalysis 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 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 blank run.



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 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.

Serum Screening

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).

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.

LC-MS Analysis

Fractions from the regions indicated in Figure 3 were collected, prepared, and enzymatically digested with trypsin (Table 1). An aliquot was removed from each sample digest and analyzed by LC-MS. Protein identifications for the chromatographic fraction at time range of 19 to 21 min (Figure 3) are listed in Table 3. These results confirm obvious differences in proteins between the samples as indicated in the bracketed region of the chromatogram, amplified in the inset. The cortisol-deficient serum sample had no complement H or apolipoprotein H, while the high rheumatoid factor serum showed a relative loss in complement H, as noted by the lower number of spectra found in comparison with the control serum.

Table 3. Protein Identifications from Fractions of Each Serum in the Time Range of 19-21 Min

A similar analysis of the chromatographic fraction from 29 to 32 min, also distinguished by differences in UV absorbancy (Figure 3), was performed on the three samples (Table 4). The bracketed region of the chromatogram shows a difference in protein levels between the control serum and the abnormal sera. The high rheumatoid factor serum has a lower level of complement component 3 compared to the control or cortisol-deficient serum. Both cortisol-deficient and high-rheumatoid-factor serum show a decreased level of complement component 4A, while cortisol-deficient serum is decreased in apolipoprotein A-1 protein levels. Several of these proteins are found at reduced levels in the sera of patients with systemic lupus erythematosus, rheumatoid arthritis, and scleroderma.2 The same proteins were identified after immunodepletion by mRP-C18 fractionation and screening comparisons of the chromatographic UV traces of suspect and control sera prior to confirmation by LC-MS.

Table 4. Protein Identifications from Fractions of Each Serum in the Time Range of 29-32 Min


The mRP-C18 column optimizes fractionation of human serum proteins after depletion of high-abundance proteins that interfere with the detection of low-abundance protein components of the plasma proteome. The ability to integrate immunodepletion and fractionation permits high recovery, excellent resolution, and reproducibility in reducing sample complexity for biomarker research while accelerating analytical throughput. The greater than 98% protein recovery from the immunodepleted serum makes it possible to validate the characterization and comparison of serum samples through differential expression of biomarkers.

In addition, reproducible protein fractionation with high resolution provides the means for rapid screening of samples by UV absorbancy, reducing sample complexity, and permitting a more effective targeted selection of samples for subsequent LC-MS analysis. Application of this technique may extend beyond sera fractionation and could prove useful in other biologic sample investigations.

William Barrett is a product manager at Agilent Technologies, 2850 Centerville Road, Wilmington, DE 19808-1610, 302.633.8120, fax 302.993.5949, bill_barrett@agilent.com

James Martosella is an R&D scientist at Agilent, James_martosella@agilent.com

Nina Zolotarjova is an R&D scientist at Agilent, Nina_zolotarjova@agilent.com

Liang-Sheng Yang is a manufacturing/quality engineer at AgilentLiang-sheng_yang@agilent.com

Cory Szafranski is a product manager at Agilent, Cory_szafranski@agilent.com

Gordon Nicol is an R&D scientist at Agilent, Gordon_nicol@agilent.com


1. Bailey J, Zhang K, Zolotarjova N, Nicol G, Szafranski C. Removing high-abundance proteins from serum,

Genetic Engineering News

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2. Grant SF, Kristjansdottir H, Steinsson K, Blondal T, Yuryev A, Stefansson K, Gulcher JR. Long PCR detection of the C4A null allele in B8-C4AQ0-C4±-DR3, J. Immunol. Methods 2000; 244:41-7.