title: Three-minute method for amino acid analysis by UHPLC and high-resolution quadrupole orbitrap mass spectrometry.
abstract: Amino acid analysis is a powerful bioanalytical technique for many biomedical research endeavors, including cancer, emergency medicine, nutrition and neuroscience research. In the present study, we present a 3 min analytical method for underivatized amino acid analysis that employs ultra high-performance liquid chromatography and high-resolution quadrupole orbitrap mass spectrometry. This method has demonstrated linearity (mM to nM range), reproducibility (intra-day <5 %, inter-day <20 %), sensitivity (low fmol) and selectivity. Here, we illustrate the rapidity and accuracy of the method through comparison with conventional liquid chromatography-mass spectrometry methods. We further demonstrate the robustness and sensitivity of this method on a diverse range of biological matrices. Using this method we were able to selectively discriminate murine pancreatic cancer cells with and without knocked down expression of hypoxia-inducible factor 1α; plasma, lymph and bronchioalveolar lavage fluid samples from control versus hemorrhaged rats; and muscle tissue samples harvested from rats subjected to both low-fat and high-fat diets. Furthermore, we were able to exploit the sensitivity of the method to detect and quantify the release of glutamate from sparsely isolated murine taste buds. Spiked in light or heavy standards ((13)C6-arginine, (13)C6-lysine, (13)C 5 (15) N2-glutamine) or xenometabolites (5-fluorouracil) were used to determine coefficients of variation, confirm linearity of relative quantitation in four different matrices, and overcome matrix effects for absolute quantitation. The presented method enables high-throughput analysis of low-abundance samples requiring only one percent of the material extracted from 100,000 cells, 10 µl of biological fluid, or 2 mg of muscle tissue.
authors.affiliation: Department of Biochemistry and Molecular Genetics, University of Colorado Denver Health Sciences Center, Anschutz Medical Campus, 12801 East 17th Ave, Aurora, CO, USA.
authors.forename: Travis
authors.initials: T
authors.lastname: Nemkov
authors.affiliation: Department of Biochemistry and Molecular Genetics, University of Colorado Denver Health Sciences Center, Anschutz Medical Campus, 12801 East 17th Ave, Aurora, CO, USA.|Metabolomics Core, Mass Spectrometry Shared Resource, University of Colorado Denver, Anschutz Medical Campus, Aurora, CO, USA.
authors.forename: Angelo
authors.initials: A
authors.lastname: D'Alessandro
authors.affiliation: Department of Biochemistry and Molecular Genetics, University of Colorado Denver Health Sciences Center, Anschutz Medical Campus, 12801 East 17th Ave, Aurora, CO, USA. kirk.hansen@ucdenver.edu.|Metabolomics Core, Mass Spectrometry Shared Resource, University of Colorado Denver, Anschutz Medical Campus, Aurora, CO, USA. kirk.hansen@ucdenver.edu.
authors.forename: Kirk C
authors.initials: KC
authors.lastname: Hansen
mesh_terms: D000596:Amino Acids / Q000032:analysis* / Q000378:metabolism*; D000818:Animals; D018893:Bronchoalveolar Lavage; D002851:Chromatography, High Pressure Liquid; D006470:Hemorrhage / Q000378:metabolism*; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000378:metabolism*; D008196:Lymph / Q000378:metabolism*; D013058:Mass Spectrometry; D051379:Mice; D051381:Rats; D017207:Rats, Sprague-Dawley; D013650:Taste Buds / Q000378:metabolism*
chemical_list: D000596:Amino Acids; C497444:Hif1a protein, rat; D051795:Hypoxia-Inducible Factor 1, alpha Subunit
keywords: Amino acids; Biofluid; Cell extract; Mass spectrometry; PLS-DA; QExactive; Tissue extract