Absolute quantification quantitative analysis

The use of isotope-labelled synthetic peptides as IS was proposed for the absolute quantification of proteins in protein expression studies [113]. If necessary, these synthetic peptides can be covalently modified to be applied as IS in for instance phosphopeptide quantification. This approach was applied in the quantitative analysis of two glutathione 5 -transferase isoforms in human liver cytosol by LC-MS in SRM mode [114]. A series of pilot experiments were performed to select the most suitable IS peptides for four human plasma proteins (hemopexin, ocl antichymotrypsin, interleiddn-6, and tumor necrosis factor-oc) [115]. Rabbit polyclonal antibodies were raised against these selected peptides and immobilized on POROS supports. These lAC columns were applied to achieve a 120-fold emichment of the antigen peptide from digested human plasma proteins. The peptides and its IS were measured by LC-MS in SIM or SRM mode. The methods appears to be a tailor method for targeted protein analysis. 

Quantitative analysis is performed to provide absolute quantification or relative quantification. In absolute quantification, the aim is to determine the amount or concentration of the analyte in absolute terms (i.e., in terms of per unit mass or per unit volume of the sample). In contrast, the aim of relative quantification is to determine the amount or concentration of the analyte relative to another analyte or of the same analyte relative to another sample. 

The field of gene expression analysis has only recently become of broader interest to users of mass spectrometry. The reason for this lag in interest was simply the lack of methods allowing the absolute quantification of NAs in general, or mRNA in particular. Mass spectrometry only allows an endpoint analysis, and even then a standard for normahzation (such as a second allele) is required. Hence, the approaches used have focused on the semi-quantitative analysis of allelic expression which, as described above, is a rather narrow-albeit expanding-field of research. 

In order to obtain quantitative results by HS-GC, the system must be calibrated. Absolute quantitation is not possible. Quantification can be done by the conventional external calibration method with liquids containing the analytes concerned in known concentrations or by means of standard addition. Pausch et al. [958] have developed an internal standard method for solid headspace analysis of residuals in polymers in order to overcome the limitations of external standardisation cfr. Chp. 4.2.2 of ref. [213a]). Use of an internal standard works quite well, as shown in case of the determination of residual hydrocarbon solvent in poly(acrylic acid) using the solid HS-GC-FID approach [959]. In the comparison made by Lattimer et al. [959] the concentrations determined by solid HS-GC exceeded those from either solution GC or extraction UV methods. Solid HS-GC-FID allows sub-ppm detection. For quantitative analysis, both in equilibrium and non-equilibrium conditions, cfr. ref. [960]. Multiple headspace extraction (MHE) has the advantage that by extracting the whole amount of the analyte, any effect of the sample matrix is eliminated the technique is normally used only for method development and validation. 

Obviously, the combination of element labeling and then detection by element mass spectrometry is still in its infancy for quantitative proteomics. However, this is such a promising method for metallomics and metallopro-teomics, because of the unique advantages (1) results can be validated due to the traceability of ICP-MS measurement (2) multiplexed analysis is easy to implement because of the availability of so many isotopes for labeling and (3) absolute quantification at a very low concentration allows comparison between the results from different laboratories. 

P NMR spectra provide quantitative information on the relative concentrations of phosphorylated metabolites. For spectra acquired under fully relaxed conditions, the area of the P NMR resonance is proportional to the concentration of the metabolite. Absolute quantifications are more difficult to perform but can also be obtained if the concentration of one of the phosphorylated metabolites in the sample (normally ATP) is measured by an independent method, such as enzymatic analysis. Unfortunately, the resonances from the a and p phosphates of ADP overlap under in situ conditions with those from the a and Y phosphates of ATP, thus precluding direct quantification of ADP in situ. The concentration of ADP in situ can be estimated indirectly from the difference in area between the a ATP or y ATP peaks and the area of the P ATP peak, which is almost exclusively derived from ATP. 

As XRF is not an absolute but a comparative method, sensitivity factors are needed, which differ for each spectrometer geometry. For quantification, matrix-matched standards or matrix-correction calculations are necessary. Quantitative XRF makes ample use of calibration standards (now available with the calibrating power of some 200 international reference materials). Table 8.41 shows the quantitative procedures commonly employed in XRF analysis. Quantitation is more difficult for the determination of a single element in an unknown than in a known matrix, and is most complex for all elements in an unknown matrix. In the latter case, full qualitative analysis is required before any attempt is made to quantitate the matrix elements. 

The reaction mechanism of this system involved the transfer of phases across the solid liquid interface. Hence, quantification using Equation (22) produced values that were overestimated. To determine the absolute phase abundances, powdered diamond was selected as an inert internal standard and was weighed into the starting solids. Acid was then added to this mixture and the standard concentration taken as its weight fraction of the sample in its entirety, i.e., solids and liquids in total. For each dataset the results of the quantitative phase analysis were adjusted according to the known amount of standard present in the system [Equation (16)]. This allowed the determination of variation in the amorphous content of the system to be assessed via Equation (17)] as well as the formation and consumption of crystalline phases. The amorphous content... 

Quantification of Calcium Sulfate Phase Transformations In order to quantitatively describe the progress of phase conversion ratios of absolute intensities of characteristic peaks of each phase s XRD pattern were compared. This method is generally considered suitable for quantitative phase analysis of mixtures [35]. To aid phase quantification, calibration curves based on prepared mixtures with known composition were used and the respective intensity ratios calculated. The intensity ratios were calculated from the intensity of peaks at 26 29.10725.43°, 29.71°/25.43° and... 

Quantitative determinations will be precise only if full corrections for losses occurring during extractions and possible detector instabilities are made. The mass spectrometer measuring the mass of molecules, the use of auxins labeled with stable isotopes provides almost ideal IS for accurate quantifications. The stable isotope dilution method [22] consists in measuring by Multiple Ion Monitoring (MIM) the ratio of normal to heavy isotopes added at the beginning of the analysis. The absolute amount of auxin in plant extract is then obtained [see 19]. 

Fischer et al. [101] investigated the simultaneous quantification of the content of several additives in PVC with an in-line diffuse reflectance probe. The signal from diffuse reflectance can be affected by a number of physical properties of the sample, rather than just its chemical make up. This makes obtaining quantitative data very difficult. Chemometric analysis showed the possibility of detecting even small amounts of additives (3%) with an absolute prediction error of 0.3%. Step-scan PA-FTIR spectroscopic studies were used to study surfactant exudation and film formation in PS-nBA latex films [102]. 

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