Assays Based on Mass Spectrometry

1 Directed Evolution of a Lipase for Desymmetrization of meso-l,4-Diacetoxycyclopentene [33] 

Isotope labeling forms the basis of a different MS-based ee-assay (92,93). It involves a fundamentally new way to measure enantiopurity. The method has been parallelized for high throughput and used a number of times in the directed evolution of enantioselective enzymes (46,64,94-96). In this MS-based approach. 

This screening system has also been applied successfully in the directed evolution of enantioselective EHs acting as catalysts in the kinetic resolution of chiral epoxides 95,96) (Section IV.A.4). Moreover, the firm Diversa has applied the MS-based method in the desymmetrization of a prochiral dinitrile (l,3-dicyano-2-hydroxypropane) catalyzed by mutant nitrilases 46). In this industrial application, one of the nitrile moieties was labeled selectively with as in N-17, which means that the two pseiido-eaaniiovaenc products (S)- N-18 and (J )-18 differ by one mass unit. This is sufficient for the MS system to distinguish between the two products quantitatively 46). 

It is emphasized that in the case of kinetic resolution, the MS measurements must be performed in the appropriate time window (near 50% conversion). If this is difficult to achieve due to different amounts or activities of the mutants being screened in the wells of microtiter plates, the system needs to be adapted in terms of time resolution. This means that samples for MS evaluation need to be taken as a function of time. Finally, it is useful to delineate the possibility of multi-substrate ee screening using the MS-based assay, which allows for enzyme fingerprinting with respect to the enantioselectivity of several substrates simultaneously. 

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Irth H Continuous-flow systems For ligand binding and enzyme inhibition assays based on mass spectrometry, chapter 5. 

Continuous-flow Systems for Ligand Binding and Enzyme Inhibition Assays Based on Mass Spectrometry... 

Continuous-flow Enzyme Assays Based on Mass Spectrometry 195 4 4 4... 

Continuous-flow Ligand Binding Assays Based on Mass Spectrometry 201... 

Niessen, H. Irth On-line coupling of high-performance liquid chromatography to a continuous-flow enzyme assay based on electrospray ionization mass spectrometry. Anal Chem 2004, 76, 3155-3161. 

Aptamer-ligand interactions can be detected by conventional assay based on, e.g., radioactive labeling, chromatography, capillary electrophoresis and mass spectrometry. These methods have been reviewed recently by Tombelli et al. [5]. Novel approach in detection of aptamer-ligand interaction is connected with aptasensors. In aptasensors,... 

Napoli et al. (23) developed a sensitive assay based on negative chemical ionization mass spectrometry to quantify retinoic acid in human plasma. Endogenous levels of all trans retinoic acid in plasma were 4.9 ng/ml, using a 0.1 ml sample. The limit of detection was less than 1 ng/ml. Direct quantification of 13-cis retinoic acid was impossible due to the inability of the GC to resolve the isomers. Barua and Olson (33) described a method to quantify all trans retinoic acid in serum using reverse phase HPLC. They detected 1.8 ng/ml of the all trans isomer, using a 2 ml serum sample and a non-acidic extraction procedure. 

TV Olah, JD Gilbert, A Banish, TF Greber, DA McLoughlin. A rapid and specific assay, based on liquid chromatography-atmospheric pressure chemical ionization mass spectrometry, for the determination of MK-434 and its metabolites in plasma. J Pharm Biomed Anal 12 705—712, 1994. 

Bogialli S, D Ascenzo G, Di Corcia A, Lagana A, Nicolardi S, A simple and rapid assay based on hot water extraction and liquid chromatography-tandem mass spectrometry for monitoring quinolone residues in bovine miUc, Food Chem. 2008 108 354-360. 

Rychlik, M., 2003. Pantothenic acid quantification by a stable isotope dilution assay based on liquid chromatography-tandem mass spectrometry. Analyst. 128 832-837. 

Rychlik, M., Netzel, M., Pfannebecker, I., Frank, T., and Bitsch, I., 2003. Application of stable isotope dilution assays based on liquid chromatography-tandem mass spectrometry for the assessment of folate bioavailability. Journal of Chromatography B. 792 167-176. 

Additionally it has been our experience that mass spectrometry as a routine detection/identification technique for bacteria is not well received by microbiologists and clinicians who prefer less expensive, less complicated approaches to bacterial typing and identification, such as methods based on polymerase chain reaction (PCR) and enzyme-linked immunosorbent assays (ELISA). For that reason we have adapted our MS approach to serve as a means of biomarker discovery that feeds candidate proteins or leads into development as PCR targets or other immunoassay techniques. 

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