Web Date: September 21, 2011
Fast, Reliable Tool Characterizes Antibodies
Small molecules aren’t the only tools drug makers have to target the protein culprits of disease. Antibodies can bind these unruly proteins, to either shut them down or to recruit the body’s immune system to attack the diseased cells. As a result, chemists have turned to so-called therapeutic antibodies more and more to treat diseases such as autoimmune disorders and cancer. Now researchers report a method using mass spectrometry to help drug developers rapidly and reliably test these antibodies (Anal. Chem., DOI: 10.1021/ac2007366).
When developing a therapeutic antibody, chemists want to know if it is the ideal match for their protein target, called an antigen. Researchers want to understand where along the antigen’s structure the antibody binds, to ensure that the bound portion is unique and not present on other proteins. If that so-called epitope isn’t unique, the antibody could cause undesirable side effects, says Michael L. Gross, a professor of chemistry, immunology, and medicine at Washington University in St. Louis.
One of the techniques scientists rely on to map epitopes is called hydrogen-deuterium exchange. In it, researchers observe how hydrogen atoms of the antibody-antigen complex swap with deuterium atoms in a deuterated solvent. Because the antibody protects the epitope from the solvent, its hydrogen atoms don’t exchange readily with the deuterium atoms. Researchers then detect which section of the antigen accumulated the fewest deuteriums. However, the method requires long incubation times--up to a couple of hours--and the deuterium-for-hydrogen exchange is reversible, so errors can appear in the epitope map.
For a faster, more reliable technique, Gross and his team use laser pulses to generate reactive hydroxyl radicals in the presence of the antibody-antigen complex. The radicals react with the side chains of amino acids exposed to the solvent and add hydroxyl groups. As in hydrogen-deuterium exchange, the antibody protects the epitope, so the radicals are less reactive toward side chains in that region. The researchers next cut up the antigen with enzymes and analyze its fragments using mass spectrometry. They then piece together a map of the antigen’s epitope by identifying which regions have the fewest hydroxyl modifications.
To prove the technique works, the research team mapped the epitope of the serine protease thrombin. Their map matched a previously published one from hydrogen-deuterium exchange (Protein Sci., DOI: 10.1110/ps.4670102).
Gross points out that the hydroxyl radicals produce irreversible modifications with reaction times so fast that the researchers don’t have to wait to do the mass spectrometry step. Also, he says, because an added hydroxyl is much heavier than an added deuterium, it’s easier to detect.
Siu Kwan Sze of Nanyang Technological University, in Singapore, points out that hydrogen-deuterium exchange must use deuterated solvents, which makes the method incompatible with proteins that don’t fold in solution. Meanwhile, the new radical method can map antigen epitopes in their native environments such as in lipid membranes and on the surfaces of cells.
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