CHAPTER 8: Group-specific Internal Standard Technology (GSIST) for Mass Spectrometry-based Metabolite Profiling

Beyond the difficulty of identifying the myriad of metabolites in even the simplest biological system, there is an increasing need to simultaneously quantify changes in the concentration and flux of these components as organisms develop and respond to environmental pressures. Although recent advances in separation technology and mass spectrometry afford 2-4-dimensional fractionation with vast improvements in the ability to ionise, detect, fragment and identify a thousand or more analytes in a single LC/MS run, matrix suppression of ionisation presents a major problem in quantification. To overcome these issues, an in vitro labelling method called ‘Group Specific Internal Standard Technology’ (GSIST) has been developed. This method isotopically codes large numbers of metabolites simultaneously with the same functional group(s) and precludes the need to synthesise metabolites of complex structure. Structurally identical, but isotopically distinct forms of a labelling agent are used to code experimental and control samples. After derivatisation, the samples are mixed and analysed in a single LC-MS run. Each metabolite from the control sample serves as an internal standard for corresponding isotopically distinct components in the experimental sample. Uses of GSIST in absolute quantification of metabolites, metabolic fingerprinting of bio-active compounds and structural identification is presented and discussed.