Making Biomarkers More Visible for Proteomics Research

Blood reaches almost every tissue in the human body, transporting essential molecules to the cells and carrying away metabolic byproducts and other substances. Some of these other substances are proteins that may be biological markers of disease. During a heart attack, for example, damaged and dying cells dump their contents into the bloodstream. The resulting concentration of the protein creatine-kinase MB is proportional to the amount of heart muscle that has been damaged.

Much of today's proteomics research is dedicated to finding additional protein-based biomarkers. However, these are difficult to measure because they are obscured by six highly abundant proteins carried in human serum or plasma. Removing the high-abundance proteins from serum samples can increase the visibility of low-abundance proteins and enable precise analysis of possible biomarkers.

Working with limited dynamic range

Some scientists believe human serum contains every protein produced by the body. To date, however, conventional measurement methods have identified just a few hundred of the estimated thousands of proteins carried in the bloodstream. One limiting factor is "dynamic range" or the ability to discern low-abundance proteins in the presence of the six highly abundant proteins that represent roughly 90% of the total protein mass in human serum.

Researchers typically measure serum proteins using methods such as two-dimensional gel electrophoresis (2DGE) and two-dimensional high-pressure liquid chromatography (2D HPLC). 2DGE can detect proteins to 1 part in 10,000 or a dynamic range of 10⁴. 2D HPLC, when used in combination with mass spectrometry (MS), has a dynamic range of about 10⁵. Neither method provides sufficient dynamic range when the six dominant proteins are present: the concentration of albumin—the most abundant protein—is about 10 billion (10¹⁰) times greater than interleukin-6, a signaling protein from the immune system.

To enable measurements within the available dynamic range, researchers can remove the high-abundance proteins from serum samples using "affinity" technologies that attract and bind one protein at a time. This improves the situation, but conventional affinity technologies typically provide incomplete removal of high-abundance interferences and leave many low-abundance proteins undetectable.

Removing multiple proteins simultaneously

The multiple affinity removal column uses immobilized antibodies to capture six high-abundance proteins from human serum samples

To overcome these problems, the Agilent multiple affinity removal system binds and retains six highly abundant proteins—albumin, IgG, IgA, transferrin, antitrypsin and haptoglobin—in one step. It does this by using affinity-purified polyclonal antibodies and optimized buffers in a convenient LC-column format.

As a sample flows through the system, the column removes the high-abundance proteins and effectively increases the concentration of low-abundance proteins. Two fractions are recovered and pooled selectively: low-abundance serum proteins flow through the column unretained and high-abundance serum proteins are eluted from the column in a separate fraction.

The optimized buffers were chosen to minimize the binding of low-abundance proteins to the dominant six and the column packing material. (Most products suffer from non-specific binding of proteins, which removes many low-abundance proteins along with the high-abundance proteins.) The buffers also prolong the lifetime of the columns, allowing regeneration and reuse for at least 200 injections.

When used with the Agilent 1100 series HPLC, the multiple affinity removal system ensures run-to-run consistency and the ability to automate sample processing. This combined solution unites the specificity of antibody-antigen recognition with the efficiency of standard LC instrumentation.

Measuring low-abundance proteins

After performing numerous experiments with the multiple affinity removal system, Agilent scientists have summarized their results in a series of application notes. Three notes describe the use of different methods to measure recovered proteins:

• "HPLC Solution for the Removal of High-Abundance Proteins in Human Serum" highlights use of the columns with the Agilent 1100 series HPLC and subsequent protein analysis using the Agilent 2100 bioanalyzer and Agilent Protein 200 Plus assay.
• "Removal of Multiple High-Abundant Proteins from Human Serum for Proteomics Sample Preparation" presents 2DGE measurements.
• "Agilent Multiple Affinity Removal System for the Depletion of High-Abundant Proteins from Human Serum—A New Technology from Agilent" shows SDS-PAGE measurements.

These cases produced four consistent results: (1) substantial removal of high-abundance proteins, (2) increased loading of low-abundance proteins onto gels and LC/MS instrumentation, (3) enhanced detection of low-abundance human serum proteins and (4) increased sample throughput.

Another note, "Binding Capacity Assessment and Optimization for the Multiple Affinity Removal System," describes the use of several column configurations and flow rates to increase low-abundance protein yields for loading onto gels or MS instrumentation.

These experiments show that the multiple affinity removal system enables greater visibility of proteins and, in turn, may yield more identifiable biomarkers for proteomics studies. With tools like this, researchers can develop a more complete portrait of the proteins carried in the bloodstream—and create new opportunities to improve human health.

For more information

To learn more about related solutions from Agilent, please see the LC & LC/MS and Proteomics sections of our Web site. For additional information about these and other Agilent life sciences products and resources, please visit the Life Sciences/Chemical Analysis main page.