Just published online is a very interesting PNAS paper from Profs. Chi-Huey Wong and Gary Siuzdak at Scripps describing their work in developing enzyme activity assays using MS detection. Specifically, they have extended the scope of the nanostructure-initiator MS (NIMS) technique which Prof. Siuzdak and others first reported in a Nature letter last year.
In that report, the authors introduced NIMS as an alternative to other MS techniques such as MALDI, ESI-ToF, or DIOS. In NIMS, nano-pores are used to trap an initiator. Analytes are then adsorbed onto the NIMS surface which is then subjected to laser irradiation. The rapid surface heating from the laser violently vaporizes the initiator and results in the ionization of the analytes. (The SEM pictures look like something from level 9 of Missile Command.) Additionally, the ionization need not be triggered by laser irradiation, but can also be conducted using ion irradiation. The researchers were able to analyze a variety of materials, including cells, tissue samples, peptides, and biofluids using NIMS. The initiators that seemed to perform the best were fluorinated or perfluorinated siloxanes.
Now that’s already pretty exciting stuff, but now they’ve taken advantage of the perfluorinated siloxane as not only an initiator, but also as a fluorous media for the immobilization of fluorous tagged materials. A fluorous tagged substrate, in this case a carbohydrate, was immobilized in the fluorous initiator filled nano-pores, exposed to an enzyme, and the products of the reaction then detected by NIMS. They have termed this technique a Nimzyme assay. The Nimzyme assay was compared with other enzyme assay methods and found to have much higher signal-to-noise ratios than either colorimetric or fluorescence methods with sensitivity greater than colorimetric methods and comparable to fluorescence. The work described not only uses NIMS as a novel detection method, but also builds on the Prof. Siuzdak’s previous work in fluorous DIOS applications and the research of Prof. Pohl and others in fluorous microarrays. One of the more important advancements here is that this is the first example of an enzyme assay on a fluorous surface. All other previous examples on fluorous microarrays have detected protein binding to immobilized substrates. They also demonstrate that cell lysates can be used directly to measure enzyme activity levels and also described enzyme inhibition by the addition of inhibitors to the cell lysates followed by incubation with the Nimzyme surfaces.
As the authors note this type of assay naturally lends itself to high throughput applications. It should be exciting to see how NIMS and fluorous NIMS develops over the next several years.