2.5.6. Amino Acid Extraction LC/MS Analysis of Isotopomer Distribution of Alanine

Dried protein hydrolysates were reconstituted by adding 300 µL of PBS and vortexing the samples, and 100 µL was transferred to a new 1.5 mL tube. Twenty-five µL of TCA (trichloroacetic acid, saturated solution, 1000 mg of TCA + 700 µL H₂O) was added and samples vortexed to mix. Samples were then centrifuged at 14,000× g for 10 min, and 50 µL were transferred to a new tube, being careful to avoid black precipitate. Then 50 µL of acetonitrile was added, and samples were mixed well by vortexing. One hundred µL of this extract was used for LC/MS analysis of alanine.

The method used to determine the isotopomers of alanine was developed by Purdue University’s Metabolite Profiling Facility, Bindley Bioscience Center, through modification of the methods used to measure amino acids. In this method, an Intrada Amino Acid column was used for the liquid chromatography (LC), followed by a quadrupole mass spectrometer (MS). Alanine is retained to ~11.5 min of the run, and the mass spectrometry returns a precursor ion of 90 m/z and a product ion of 44 m/z. The fragment of 44 m/z (with chemical formula C₂H₆N) contains four hydrogens that can potentially be replaced by deuterium during the synthesis process. The precursor (alanine, C₃H₇NO₂) and product (C₂H₆N) will increase mass equally as deuterium is added to the molecule. For this method, the LC/MS machine and software is programmed to measure the intensity/area of the peaks of molecules with precursor → product ion pair of 90 m/z → 44 m/z; 91 m/z → 45 m/z; 92 m/z → 46 m/z; 93 m/z → 47 m/z; and 94 m/z → 48 m/z; in order to measure the intensity/area of isotopomer (M) with no heavy isotopes (M0), one (M+1), two (M+2), three (M+3) and four (M+4), respectively. Supplementary Materials Information S1 shows the distribution of alanine M0, M+1, etc., in a sample from an unlabeled animal (blank) and eight samples from D₂O labeled animals, with corresponding LC/MS spectra of samples.