Stable isotopes standards

Table 9.1 A selection from the Table of Light Stable Isotope Standard Reference Materials avail-...

Anderson NL, Anderson NG, Haines LR, et al. Mass spectrometric quantitation of peptides and proteins using stable isotope standards and capture by anti-peptide antibodies (SISCAPA)./. Proteome Res. (2004) 3 235-244. 

Table 7.6 Internationally accepted stable isotope standards for hydrogen, carbon, oxygen, nitrogen, and sulfur.

Stable Isotope Standards and Capture by Anti-Peptide Antibodies... 

Immunoenrichment can be applied at the peptide level, particularly in the stable isotope standards and capture by anti-peptide antibodies (SISCAPA) method (140). Target peptides together with their stable isotope-labeled counterparts (used as quantitation references) are enriched by antibodies that are developed against antigens with almost the same sequences. The antibodies are often covalently attached to magnetic beads for sample cleanup. This technique boasts an improved limit of quantitation and increased inter-laboratory reproducibility (141). 

With BBSs, the internal standard is present in the extraction solvent and can only equilibrate with the endogenous metabolites that are extracted from the DBS. Therefore, isotope dilution principles do not apply to the extraction step since they are not in the blood (blood spot) to begin with. However, subsequent steps such as derivatization, sample transfer, and so on do indeed follow the principles of isotope dilution. To note the differences between analyzing liquid versus BBSs using stable isotope standards and the fact that quantification is less accurate using BBSs, the term pseudoisotope dilution is often used. 

The most used current method for measuring the concentration of low-abundance proteins in plasma is termed SISCAPA, which stands for stable-isotope standards and capture by antipeptide antibodies [37]. In this method, which is a hybrid of mass spectrometry and immimoassay, proteins are first digested with a protease into peptides. After digestion, isotope labeled internal standards are spiked into the sample to allow ratiometric quantitation. The mixture is then exposed to antibodies that have been raised to bind to the desired peptides more than one antipeptide antibody can be used in this step if a multiplex assay is desired. Washing steps are used to remove the excess nontarget peptides, and the peptides are then eluted from the antibodies and analyzed by LC-MS/MS. 

Cysteine conjugates and the twin-ion technique. In addition to studies of glutathione conjugates, we have utilized the twin--ion technique to study reaction mechanisms in reactive metabolite formation from acetylhydrazine and isopropylhydrazine. This technique has proven to be a useful tool when applied to biotransformation studies. Hammar and Holmstedt (15) used the naturally occurring isotopic doublet of chlorine to study the metabolism of chlorpromazine. Morfin et al. (16) were the first to use stable isotope standards in studies of the pathways of testosterone... 

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