Quantitation analysis strategies

HPLC quantitation methodologies are similar to those used in other chromatographic techniques and fall into three different categories.2,4,5 Both peak areas and peak heights can be used, though peak area methods are more popular. Readers are referred elsewhere for details and numerical calculation on these quantitation methodologies. Calculation examples are found in Chapter 6. 

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In addition to protein identification and characterization, another major goal of proteomics research is to quantify protein expression levels. However, MS is not inherently quantitative. Thus, the intensity of a peptide ion introduced to the mass spectrometer via ESI or MALDI does not reflect necessarily the amount of peptide present in the sample, due to the strong dependence of ionization on the physical and chemical nature of the analyte. To overcome this challenge, numerous quantitative analysis strategies have been developed to measure the differences in protein abundances between two different cellular states of a biological system (for example, normal and diseased cells). 

It has been demonstrated also that the iTRAQ tandem mass spectrometric quantitative analysis strategy can be used in conjunction with the quadrupole ion trap by performing multiple stages of mass analysis (that is, MS ) [125], For example, chemical derivatization with the iTRAQ reagent not only labels the N-terminus of a peptide, but the lysine side chain also. Thus, tryptic peptides with a modified lysine residue present at the C-terminus will produce a yj product ion at m/z 291 following ClD-tandem mass spectrometry. To generate the low m/z iTRAQ reporter ions required for quantitation, the yj product ion is isolated and subjected to data-dependent CID-MS. Using this approach, peptide identification is achieved in the MS/MS scan, while quantitation is achieved via MS. ... 

Because X-ray counting rates are relatively low, it typically requires 100 seconds or more to accumulate adequate counting statistics for a quantitative analysis. As a result, the usual strategy in applying electron probe microanalysis is to make quantitative measurements at a limited collection of points. Specific analysis locations are selected with the aid of a rapid imaging technique, such as an SEM image prepared with backscattered electrons, which are sensitive to compositional variations, or with the associated optical microscope. 

Precipitation reactions have many applications. One is to make compounds. The strategy is to choose starting solutions that form a precipitate of the desired insoluble compound when they are mixed. Then we can separate the insoluble compound from the reaction mixture by filtration. Another application is in chemical analysis. In qualitative analysis—the determination of the substances present in a sample—the formation of a precipitate is used to confirm the identity of certain ions. In quantitative analysis, the aim is to determine the amount of each substance or element present. In particular, in gravimetric analysis, the amount of substance present is determined by measurements of mass. In this application, an insoluble compound is precipitated, the precipitate is filtered off and weighed, and from its mass the amount of a substance in one of the original solutions is calculated (Fig. 1.6). Gravimetric analysis can be used in environmental monitoring to find out how much of a heavy metal ion, such as lead or mercury, is in a sample of water. 

Steady-state experiments can also be designed within the same kind of strategy. As an example, we can cite recent works [25,45], where the results of a quantitative analysis of the resolved absorption spectra of a number of trans and cis isomers of cyanine dyes were eompared with calculated oscillator strengths and transition energies so as to propose the identification of the observed phototropic species as well defined cis isomers. 

Ji, J. Chakraborty A. Geng, M. Zhang, X. Amini, A. Bina, M. Regnier, F. Strategy for qualitative and quantitative analysis in proteomics based on signature peptides. J. Chromatogr. B Biomed. Sci. Appl. 2000, 745,197-210. 

Zhang, H., Yan, W., and Aebersold, R. (2004) Chemical probes and tandem mass spectrometry A strategy for the quantitative analysis of proteomes and subproteomes. Curr. Opin. Chem. Biol. 8, 66-75. 

Quantitative analysis in ICP-MS is typically achieved by several univariate calibration strategies external calibration, standard addition calibration or internal standardisation. Nevertheless multivariate calibration has also been applied, as will be presented in Chapters 3 and 4. 

Internal standard (IS) calibration requires ratioing of an analytical signal to an IS which has very similar characteristics to that of the analyte of interest (an element which is similar to the analyte either in mass, ionisation potential or chemical behaviour). Quantitative analysis applying internal standardisation is the most popular calibration strategy in ICP-MS, as improvements in precision are obtained when the technique is appropriately used. Of course, the validity of this calibration method requires that one ensures a good selection of the correct internal standard. For this purpose it is possible to resort to chemometric methods [16]. 

A strategy for producing AChE biosensors for quantitative analysis of substrates and for a qualitative assessment of the OPs present has concentrated on the modification of the enzyme active site. Possible differences in the inhibition mechanism have been further identified in genetically modified AChE, principally from Drosophila melanogaster [182] structural changes and the replacement of specific amino acids within the AChE have resulted in the greater sensitivity of particular mutant AChE to specific OPs. For example, Boublik et al. [183]... 

The identification of all or most substances in one or more chromatograms, even with extensive spectroscopic data, can be a challenging and difficult process. The available data may not be sufficient to even tentatively identify all the components, especially if pure authentic samples of suspected substances are not available in the laboratory. Calibration of a broad spectrum method for quantitative analysis is delayed until the desired components are identified. For these reasons, and the general preoccupation with target analytes, the BS strategy is much less common than the target analyte strategy. 

The third stage of our strategy is discussed in Sections IX and X. Our discussion is speculative, since quantitative analysis is lacking at present. In Section IX, we point out that, in reaction dynamics, breakdown of normal hyperbolicity would also play an important role. Such cases would include phase transitions in systems with a finite number of degrees of freedom. In Section X, we will discuss the possibility of bifurcation in the skeleton of reaction paths, and we point out that it corresponds to crisis in multidimensional chaos. This approach offers an interesting mechanism for chemical evolution. 

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