Amino Acid Analysis in Physiological Samples by GC–MS with Propyl Chloroformate Derivatization and iTRAQ–LC–MS/MS

Abstract

Two mass spectrometry-based methods for the quantitative analysis of free amino acids are described. The first method uses propyl chloroformate/propanol derivatization and gas chromatography–quadrupole mass spectrometry (GC–qMS) analysis in single-ion monitoring mode. Derivatization is carried out directly in aqueous samples, thereby allowing automation of the entire procedure, including addition of reagents, extraction, and injection into the GC–MS. The method delivers the quantification of 26 amino acids. The isobaric tagging for relative and absolute quantification (iTRAQ) method employs the labeling of amino acids with isobaric iTRAQ tags. The tags contain two different cleavable reporter ions, one for the sample and one for the standard, which are detected by fragmentation in a tandem mass spectrometer. Reversed-phase liquid chromatography of the labeled amino acids is performed prior to mass spectrometric analysis to separate isobaric amino acids. The commercial iTRAQ kit allows for the analysis of 42 physiological amino acids with a respective isotope-labeled standard for each of these 42 amino acids.