MS-based quantitation methods

With the label-free methods in the repertoire of quantitative proteomics practitioners, there is still a need for label-assisted methods to perform quantitation with improved accuracy and precision. Label-assisted methods incorporate stable isotope labels into internal standards (IS) for quantitation this MS-based quantitation method is termed stable isotope dilution. The incorporation of stable isotope labels can be achieved either with or without... 

Lemeer, S., Hahne, H Pachl, F and Kuster, B. 2012. Software tools for MS-based quantitative proteotnics A brief overview. Methods Mol. Biol. 893 489-499. 

In LC-MS, specific ionisation conditions can be required for different types of species. This means that in LC-MS studies on extractable additives, it is necessary to use a range of experimental conditions to cover detection of all types of potential species. Depending on instrument type, it is also possible to isolate ions in complex matrices and obtain positive identifications by further unique fragmentation of these ions (by MS-MS or MSn). Quantitative methods based on this secondary ionisation can be employed. The mass accuracy of LC-MS detection systems continues to improve. Accurate mass measurement improves the certainty of identification. Advanced systems are typically offering 1-2 ppm (mass dependent) mass accuracy. 

With respect to the practical considerations of gas flow and vacuum requirements, the PHPMS experiment might, upon cursory consideration, appear to be easily extended into the VHP region. That is, several MS-based analysis techniques routinely use ion source pressures of 1 atm. However, when an attempt to increase the pressure within a PHPMS ion source is made, the factors that do become problematic are those related to the subtle principles on which the method is based. Most importantly, the PHPMS method requires that the fundamental mode of diffusion be quickly established within the ion source after each e-beam pulse, so that all ions are transported to the walls in accordance with a simple first-order rate law while the IM reactions of interest are occurring. This ensures that a constant relationship exists between the ion density in the cell and the detected ion signal. The rates of the IM reactions can then be quantitatively determined from the observed time dependencies of the reactant ion signal because the contribution of diffusion to the time dependencies are well known and easily accounted for. 

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). 

Warren EN, Elms PJ, Parker CE, et al. Development of a protein chip a MS-based method for quantitation of protein expression and modification levels using an immunoaffmity approach. Anal. Chem. (2004) 76 4082-4092. 

Liquid chromatography/mass spectrometry (LC/MS)-based techniques provide unique capabilities for pharmaceutical analysis. LC/MS methods are applicable to a wide range of compounds of pharmaceutical interest, and they feature powerful analytical figures of merit (sensitivity, selectivity, speed of analysis, and cost-effectiveness). These analytical features have continually improved, resulting in easier-to-use and more reliable instruments. These developments coincided with the pharmaceutical industry s focus on describing the collective properties of novel compounds in a rapid, precise, and quantitative way. As a result, the predominant pharmaceutical sample type shifted from nontrace/pure samples to trace mixtures (i.e., protein digests, natural products, automated synthesis, bile, plasma, urine). The results of these developments have been sig-... 

Interestingly, many of the current LC/MS approaches for pharmaceutical analysis are extensions of gas chromatography/mass spectrometry (GC/MS) (Foltz, 1978), mass spectrometry (Garland and Powell, 1981), and MS/MS (McLafferty, 1983)-based methods. With the introduction and widespread use of LC/MS-based methods, these fundamental approaches for quantitative and qualitative structure analysis became more routinely applicable to a wider scope of pharmaceuticals. 

At the time, this application provided a powerful benchmark for the use of LC/MS-based methods in the pharmaceutical industry. This particular assay successfully supported several clinical studies with sensitive and reliable results. This performance was bench-marked on more than 4000 clinical samples and led to a wider scope of application and the development of routine, standard methods for quantitative bioanalysis. 

Hence radical damage to DNA can be detected by the characterization and quantitative measurement of the products derived from the purine and pyrimidine bases. The method is demonstrated here by applying model systems of DNA treated with superoxide-generating systems or hydrogen peroxide in the presence of an iron chelate (Aruoma et al., 1989). Products of radical-mediated base damage in DNA are measured using GC-MS-SIM after acid hydrolysis of DNA and trimethy lsily lation. 

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