Dynamic Range is your Friend
In medical diagnostics, the benefits of profiling several proteins versus a single biomarker is that together, multiple markers can provide close to 100% sensitivity and selectivity, compared with 30% to 50% for single markers.
"In biology having just one difference between disease and normal states is unlikely," says Mary F. Lopez, Ph.D., strategic collaborations leader at PerkinElmer (www.perkinelmer.com).
A seminal article, by Petricoin and Liotta, appearing in The Lancet in 2000, was perhaps the first report demonstrating that profiling based on mass spectrometry (MS) could serve as a disease diagnostic. What was revolutionary about this work was the idea that high-abundance proteins can assist, rather than interfere with, proteomic analysis.
Carrier proteins in serum or blood, normally removed to reduce background noise, bind to lower-concentration biomarkers shed from tumors and other tissues.
"The best way to find these biomarkers is to capture the carriers and see what is bound to them," says Dr. Lopez, who described the original Petricoin and Liotta work as a "paradigm shift" despite the fact that the methods they used in 2000 were quite crude. Recently, the two researchers validated their idea by demonstrating spectral profiling and purification of ovarian cancer biomarkers captured through high-abundance carrier molecules.
However, protein profiling will not enter "prime-time" medical diagnostics markets without a significant boost in its ability to pick out protein markers at very low concentrations. "A high-resolution platform will provide confidence and statistical significance," says Dr. Lopez.
PerkinElmer demonstrated, in a recent paper on Alzheimer's disease markers, a technique for identifying biomarkers by capturing albumin on a 96-well plate, selectively eluting the markers, and analyzing them by MALDI MS. Selective elution uses a proprietary solvent that knocks the biomarker off albumin, which is retained on the plate.
A good deal of profiling employs protein (or antibody) arrays, the protein counterparts of successful gene arrays. Protneteomix (www.protneteomix.com) specializes in protein arrays (especially antibody arrays) for drug discovery based on three core competencies.
The firm's proprietary, cell-free in vitro protein expression expands the numbers and types of available, "array-able" molecules to include cytotoxic and prokaryotic proteins. This synthetic capability can generate purified, tagged proteins from any organism with a sequenced genome. Through its antibody array manufacturing, Protneteomix provides custom arrays on 3-D plane supports without chemical modification of targets. Finally, for protein interaction experiments, the company offers labeling and detection methodology operating at attomole (10-18) levels for immobilized proteins and antibodies.
Protneteomix' technology platform allows comparison of expression levels for "many proteins" in a single binding assay (for example pre- and post-treated cells), relative toxicity evaluation, and biomarker discovery for cancer and other diseases.
"Our technology has replaced many thousands of Western blots," says Vehary Sakanyan, Ph.D., professor at the University of Nantes and a principal with Protneteomix. "The benefit of very low sample consumption is especially relevant with biopsy material."
One application of antibody arrays is analyzing post-translational protein modifications, which cannot be done using DNA arrays. Here, antibodies are generated against proteins possessing specific phosphorylation patterns.
Antibody arrays are only as good as the antibodies they contain, but even the best exhibit cross-reactivity, which limits their reliability. To cut down on this phenomenon, Dr. Sakanyan suggests using only the highest-quality antibodies for spotting, depleting high-concentration proteins beforehand, and perhaps analyzing proteins from only one location in the cell.
"One might also consider replacing full-length antibodies with antibody fragments which retain high affinity and specificity, but with lower cross-reactivity," he adds.
Protein arrays have led to orders of magnitude more answers per experiment, says Brett Stillman, Ph.D., manager for microarray technologies at Whatman (www.whatman.com).
"Protein arrays also have the potential to replace more and more conventional assay technologies," he adds. Arrays, particularly antibody arrays, facilitate multiplex capture and are about as efficient as running individual ELISA assays, especially when sample volumes are limited and precious. Dr. Stillman likes the ability of protein arrays to monitor protein-protein interaction, calling them "more powerful" than co-immunoprecipitation.
Whatman's protein profiling product line includes the FAST slides, found in array products manufactured by other companies. More than 80 scientific papers cite FAST in their methods section.
Whatman's Serum Biomarker Chip, based on single antibody capture with direct fluorescent labeling of serum samples, profiles 120 of the most common serum biomarkers. The company also offers custom protein array and sample processing services in the above formats and others.
In first quarter 2006 Whatman plans to release a protein array diagnostic for the European market that will measure auto-antibodies associated with different collagenosis and vasculitis-related autoimmune diseases.
TeleChem-ArrayIt (www. arrayit.com) has provided tools for microarray DNA analysis since 1996, and for the last few years has offered protein array products plus a line of microcontact printing devices for protein microarray manufacture.
ArrayIt's protein array tools are used by more than 3,000 research groups worldwide, including many academics who were responsible for developing methods based on these tools. The precision NanoPrint Microarray system (with 60 and 210 slide capacity) and the NanoPrint Protein Edition Microarrayer are high-throughput instruments for the manufacture of protein microarrays. ArrayIt offers the SpotBot microarrayer for personal, low-throughput array manufacture.