PROTOCOL PRODUCERS Tang (from left), Min, and Mrksich developed a practical method to screen chemical libraries by using mass spectrometry. COURTESY OF MILAN MRKSICH

Use of MS to report results of screening chemical libraries against enzymatic activity has not been practical before, Mrksich says. Preparing samples is time-consuming, usually requiring chromatographic steps to clean up and enrich the analyte. Applied to large libraries, the task would be overwhelming. This hurdle could be overcome by using a protocol that requires only one rinse for sample preparation.

Knowing that self-assembled monolayers (SAMs) are well suited for analysis by matrix-assisted laser desorption-ionization time-of-flight (MALDI-TOF) MS, Mrksich and coworkers use arrays of reaction wells that are lined with SAMs on which the substrate of the enzyme of interest is immobilized. When the enzymatic reaction is over, the wells are washed once, and they're ready for MS analysis. A change in the mass of the substrate attached to the SAMs indicates enzyme activity.

"To the best of my knowledge, there is no other group combining the throughput of an array-format assay with the readout of mass spectrometry for small-molecule screening," comments David A. Eiznhamer, director of biological sciences at Advanced Life Sciences, Woodbridge, Ill.

To demonstrate the strategy, the researchers tethered a peptide corresponding to the portion of the natural substrate that is cleaved by anthrax lethal protein. Substrate in a well that contains inactive compound from a chemical library, when analyzed by MALDI-TOF MS, will show a decrease in mass after being cut by anthrax lethal factor. But in a well that contains an inhibitor that stops anthrax lethal protein from cutting the peptide, the substrate will show no change in mass. In this way, Mrksich and coworkers identified a small molecule, DS-998, that inhibits anthrax lethal factor at micromolar levels. They also demonstrated its inhibitory activity in human cells [Nat. Biotechnol., 22, 717 (2004)].

A practical MS protocol for screening chemical libraries has two major advantages, according to Mrksich. First, it eliminates the need to label assays with fluorescent or radioactive tags. The mass change itself reports the activity. And second, it opens the door to biochemical assays that are not amenable to labeling. For example, enzymes that modify carbohydrates remain difficult to assay with current strategies, he explains. The method is generally applicable, as long as the enzyme's action on a substrate involves a change in mass, he adds.