Amino Acid Analysis Methods and Protocols
Amino Acid Analysis (AAA) is an integral part of analytical biochemistry. In a relatively short time, the variety of AAA methods has evolved dramatically with more methods shifting to the use of mass spectrometry (MS) as a detection method. Another new as
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1. Introduction Comprehensive analysis of amino acids in large sample sets presents challenges due to their high water solubility, range of ionic characteristics, and lack of a universal and selective chromophore. Highperformance liquid chromatography (HPLC) coupled with pre- (1–3) or postcolumn (4) derivatization and UV/Vis or fluorescence detection has established dominance as a standard method for amino acid analysis. Gas chromatography (GC) with flame ionization or mass spectrometry (MS) detection can also be employed for separating volatile derivatives of amino acids (5, 6). However, the need for derivatization often requires complete evaporation of solvent before derivatization. The derivatization process may
Michail A. Alterman and Peter Hunziker (eds.), Amino Acid Analysis: Methods and Protocols, Methods in Molecular Biology, vol. 828, DOI 10.1007/978-1-61779-445-2_1, © Springer Science+Business Media, LLC 2012
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also compromise data quality due to inconsistent yields of a single derivative for each amino acid and derivative instability. For both HPLC and GC methods, resolving the suite of amino acids have required 20–60 min analysis time per sample, reducing their utility for analysis of large numbers of samples required for mutant screening or other large-scale projects. LC coupled with tandem mass spectrometry (LC–MS/MS) provides a specific and sensitive technique for amino acid analysis without derivatization (7–10). The method described here adapts a 96-well microtiter plate format for sample preparation using aqueous extraction (11) followed by filtration to eliminate cell debris. Twenty proteinogenic amino acids as well as γ-aminobutyric acid (GABA), hydroxyproline (Hyp), and the biosynthetic pathway intermediates anthranilate, homoserine (Hse), and S-methylmethionine (SMM) are separated by reverse-phase chromatography using the ion-pairing agent perfluoroheptanoic acid in the mobile phase in a total 6 min assay time (12). Detection and quantification are achieved using electrospray ionization/tandem mass spectrometry in multiple reaction monitoring (MRM) mode to monitor transitions of the protonated molecules to their specific product ions. Two internal standards, Val-d8 and Phe-d8, are spiked in the standards and seed samples allowing extraction normalization and loading control, respectively. The calibration curves for each compound are created by plotting amino acid standard concentrations as x-axis and peak area ratio of amino acid/Phe-d8 as y-axis using linear regression and the endogenous concentrations of the 25 compounds calculated according to the slope and intercept from the standard curves and the peak area ratio of AA/Phe-d8 in the extract. The described method is suitable for quantifying underivatized amino acids and related metabolites in Arabidopsis seed tissue with good reproducibility, high accuracy, and low intra- and interday variation. This approach may also fulfill the requirements for the diagnosis of amino acid metabolism-related disorders in clinical fields
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