Presently LC-MS is expanding beyond its traditional role at the front line of small molecule diagnostic measurements to large molecule (mainly protein) diagnostics. Previously, radioimmune assay (RIA) for metabolites and hormone levels was the approach of choice, but it has too much baggage. Shortcomings to definitive RIA performance included the presence of autoantibodies in patients’ serum, difficulty in automating cumbersome protocols, and cross reactivity of antibodies, all causing a surfeit of false positives.
With the need to assess specific proteins, a different set of issues present themselves, including the requirement for high sensitivity to analytes present in vanishingly small amounts, coupled with their miscreant behavior. Dr. Clarke consigns to the bad-behavior category, molecules that are unstable, complex with other molecules, and clean up only with difficulty. Therefore, building new diagnostic tests requires robustness, small volumes, accuracy, precision, and reproducibility.
Dr. Clarke and his associates have studied the angiotensin system in the development of a superior LC/MS assay. Angiotensin II is the final active messenger of the renin-angiotensin pathway, it binds to a specific receptor, precipitating vasoconstriction and fluid retention, leading to an increase in blood pressure.
Since many drugs developed to control hypertension regulate blood pressure at various points in the renin-angiotensin system, it is critical to have accurate measures of the members of this cascade.
Dr. Clarke described his group’s efforts to improve the preparative steps, automating them, and lowering the volumes of material required. The group found that LC/MS/MS assays required much less sample volume and were more specific than traditional RIA. One of the major problems of RIA that the group has resolved is its limited dynamic range, which extends from 0.65 ng/mL/hr to 14 ng/mL/hr. Dr. Clarke reported that his group’s LC/MS/MS procedure improved sensitivity by an order of magnitude.
“We need to face new problems with protein analysis that do not exist in small-molecule assays,” he said, “and external testing agencies need to be educated with regard to these new methodologies.”
Ovarian Cancer Antigens
“There are several strategies for the early detection of cancer,” says Michael Tainsky, Ph.D., professor at Wayne State University School of Medicine. “One can screen patients’ serum for plasma DNA, antigenic proteins, marker proteins, or for antibodies.”
This range of options opens up the possibility of developing a number of different detection protocols. It is unlikely that any single protein can serve as an unambiguous marker for malignancy, therefore effective detection systems will require that a number of different proteins be used. Thus, a definitive diagnosis would represent the composite value of several cancer-related markers.
Dr. Tainsky’s program harnesses antibodies in patients’ serum for the detection of cancer-specific epitopes using peptides selected for IgG binding from phage-display cDNA libraries. This detection method is limited to amino acid epitopes and cannot detect epitopes produced through post-translational modifications such as those involving glycosylation or addition of lipid molecules to the original amino acid chain.