Proteins isotope dilution analysis

Giusti, P., Schaumloffel, D., Encinar, J. R., and Szpunar, J., Interfacing reversed-phase nanoHPLC with ICP-MS and on-line isotope dilution analysis for the accurate quantification of selenium-containing peptides in protein tryptic digests. Journal of Analytical Atomic Spectrometry 20(10), 1101-1107, 2005. 

P Felker. A gas-liquid chromatographic-isotope dilution analysis of cysteine, histidine, and tryptophan in acid-hydrolyzed protein. Anal Biochem 76 192-213, 1976. 

Radioimmunoassay (RIA) is a technique based on the formation of antigen-antibody complex. This technique essentially involves the application of isotope dilution analysis. An antigen is typically a protein of molecular weight greater than 10,000 that stimulates the production of antibody in an animal body. The antigen subsequently binds with the antibody. 

Oven/iew Waters, Sediments, and Soils. Ion-Selective Electrodes Water Applications. Isotope Dilution Analysis. Liquid Chromatography Size-Exclusion Liquid Chromatography-Mass Spectrometry Mass Spectrometry Peptides and Proteins. Voltammetry Overview. 

Isotope Dilution Analysis in the Quantitative Study of Proteins... 

In recent years, a remarkable trend can be observed i.e. the utilization of inductively coupled plasma-mass spectrometry (ICP-MS) as an attractive complement for protein quantification. If the heteroatom-containing proteins are known and the standards are available, the absolute quantification of proteins can be easily obtained via element analysis by ICP-MS. However, when ICP-MS is interfaced to a separation system, such as HPLC, the organic solution and inorganic salts introduced into ICP-MS usually decrease the instrumental stability and detection limits. To overcome the problems, isotope dilution analysis is introduced to an ICP-MS-based linked system. ... 

Isotope dilution analysis for protein quantification with ICP-MS is usually performed by linked techniques based on the coupling to an effective separation method, such as HPLC or CE. Generally, proteins to be analyzed should contain ICP-MS-detectable elements, including both naturally occurring and artificially labeled. After introduction of the spike that has different isotopic composition from the analyte, the elemental isotope ratio can be measured in ICP-MS and thus the detectable elements can be absolutely quantified by use of the isotope dilution equation. If proteins have been identified with biological mass spectrometry, or the elemental stoichiometric ratios of proteins have been known, the amount of proteins can be deduced from the elemental concentration of the proteins. 

Harrington et al. labeled a copper-containing protein rusticyanin (Rc) with Cu for use as a spike material in species-specific isotope dilution analysis, and... 

Later, Busto et al. used synthesized Fe-labeled transferrin to determine individual transferrin isoforms in human serum samples for the species-specific mode. The stability of the prepared proteins was tested for 1 week and no iron exchange had occurred. They concluded that the results are in good agreement with other calibration methods, e.g. the species-unspecific method however, the species-spedfic mode can offer better precision. Hoppler et al. also synthesized and characterized Fe-labeled Phaseolus vulgaris ferritin for isotope dilution analysis. " ... 

Muniz et al. quantitatively analyzed the metal containing proteins in human serum by anion-exchange chromatography coupled to post-column isotope dilution analysis with double focusing ICP-MS. Parallel analysis of human serum from healthy volunteers and patients on hemodialysis was also undertaken. They found different amounts of iron-containing proteins between healthy and renal disease individuals and indicated that Fe bound to albumin decreased in patients with renal disease. ... 

Selenium Speciation in Edible Animal Tissues Reports on Se specia-tion analysis in edible animal tissues have been scarce. Speciation analysis of Se in cod muscle tissue was performed by separating the species using both RP- and SE-HPLC prior to ICP-MS detection. The main Se compound found in enzymatic hydrolysates was selenomethionine [42], This selenocompound was absent in MeOHDHCl extracts, indicating that Se was mainly incorporated into proteins. A number of unidentiFed Se species were also detected in cod muscle tissue, the separated Se compounds being quantised on-line by post-column isotope dilution [42], Soluble Se compounds extracted from muscles of chicken, turkey, duck, ostrich, lamb, cattle, and pig were separated by SEC with ICP-MS detection. Four peaks were observed, but distribution of Se among these peaks varied considerably in tissues from different animal species [86]. 

Isotope dilution in combination with the substoichiometric principle is applied in various ways. The most important examples are radioimmunoassay for protein analysis and DNA analysis. In radioimmunoassay, radionuclides are used as tracers and immunochemical reactions for isolation. Radioimmunoassay was first described in 1959 by Yalow and Berson, and since then has found very broad application in clinical medicine, in particular for the measurement of serum proteins, hormones, enzymes, viruses, bacterial antigens, drugs and other substances in blood, other body fluids and tissues. Only one drop of blood is needed, and the analysis can be per-fonned automatically. Today more than 10 immunoassays are made annually in the United States. The most important advantages of the method are the high sensitivity and the high specificity. In favourable cases quantities down to 10 g can... 

A stable isotope dilution assay using mass spectrometry to measure insulin, proinsulin, and C-peptide has been developed. The difference in mass among the three analytes allows specific measurement of each protein. Comparison of patient samples revealed that most, but not all, results were higher by immunoassay than mass spectrometry. Thus immunoassays may overestimate insulin, particularly at low concentrations. The high protein concentration in the serum requires extraction of proteins (e.g., by immunoaffinity) and purification by high-performance liquid chromatography (HPLC) before quantification by mass spectrometry. This method is not suitable for routine laboratory analysis. 

A dramatic selective enrichment in nitrotyrosine content was observed within apoA-I recovered from serum and human atherosclerotic lesions. The analysis of serum from sequential subjects demonstrates that the nitrotyrosine contents of apoA-I were markedly higher in individuals with cardiovascular disease. An increase in nitrotyrosine was observed in the atherosclerotic lesions and plasma of apolipoprotein A-I-deficient mice (Parastatidis et al., 2007). LC MS/MS studies confirm that nitration of apoA-I occurs specifically on tyrosine-192 (Shao et al., 2005). Similarly, LC—MS/MS methods were used to identify tyrosine residues 292 and 422 at the carboxyl terminus of the 3 chain as the principal sites of fibrinogen nitration in vivo (Parastatidis et al., 2008). Stable isotope dilution LC MRM/MS methodology will likely be employed extensively in the future to assess the role of protein tyrosine nitration in the oxidative stress associated with cardiovascular disease. 

Although MS is most often used for qualitative analysis and structural determination, several methods have been developed for the quantitative assessment of proteins, through peptides or glycopeptides, in order to determine the extent of their over- or under-expression in cellular systems. Some proteomic methodologies use natural isotope incorporation to prepare internal standards for isotope dilution experiments. The protein concentration may then be determined by quantifying the abundances of either the peptides or glycopeptides. These methods were summarized and compared in a review produced in 2007 [119]. 

Protein Analysis Quantification of proteins is required in many scientific fields, such as clinical chemistry and biochemistry. Various isotope dilution strategies, relying on the use of compounds enriched in H, or 0, have been used... 

LC—MS/MS assays for 25-OHD [27] are now commonplace in clinical laboratories, especially larger reference laboratories. Often, the first step in the analysis is a liquid—liquid extraction of nonpolar 25-OHD and a deuterated internal standard from serum into an organic solvent, followed by evaporative concentration and injection into the LC—MS/MS. Additional reported metirods for specimen preparation include manual and online solid-phase extraction and simple protein precipitation with an organic solvent. The advantages of LC—MS/MS assays over traditional radioimmimoassays and more current chemiluminescent immunoassays include the use of an isotope dilution for precise quantitation, as well as the ability to distinguish and separately quantitate 25-hydroxy vitamin D2 and 25-hydroxy vitamin D3. There is considerable controversy as to which method of 25-OHD quantitation is the best, however, since many large scale population studies on vitamin D, such as the Women s Health Initiative (WHl) and the National Health and Nutrition Examination Survey (NHANES), utilized radioimmunoassays [28]. 

For metabolomics studies using IM-MS, electrospray ionization (ESI) is commonly used and the quantitative analysis will be different than the Ni. With ESI, internal standards, standard addition, isotope dilution, or external calibration curves are common practices for relative quantitative analysis. An example of ESI-IM-MS quantitative analysis was demonstrated for 20 phenylthiohydantoin (PTH)-derivatized amino acids, the final products in the Edman sequencing process of peptides and proteins. Detection limits for these amino acid derivatives ranged from 1.04 to 3.52 ng (less than 17 pmol).<" ° Quantitative analysis has not yet been established with ESI-IM-MS for applications to metabolomics. 

The main challenge when applying this method for protein analysis is the lack of commercial isotopically labeled proteins. Thus, most applications are focused on small molecules, such as organic mercury, organic tin and so on. However, there is increasing interest in the use of the ICP-MS linked system and species-specific isotope dilution for quantification of peptides or proteins due to the outstanding performance of ICP-MS. 

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