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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 1?; 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.

SUBMITTER: Nemkov T

PROVIDER: S-EPMC4624008 | biostudies-literature | 2015 Nov

REPOSITORIES: biostudies-literature

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Three-minute method for amino acid analysis by UHPLC and high-resolution quadrupole orbitrap mass spectrometry.

Nemkov Travis T D'Alessandro Angelo A Hansen Kirk C KC

Amino acids 20150610 11

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 %), sensi ...[more]

PMID: 26058356

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Project description:There is an immediate need for improved methods to systematically and precisely quantify large sets of peptides in complex biological samples. To date protein quantification in biological samples has been routinely performed on triple quadrupole instruments operated in selected reaction monitoring mode (SRM), and two major challenges remain. Firstly, the number of peptides to be included in one survey experiment needs to be increased to routinely reach several hundreds, and secondly, the degree of selectivity should be improved so as to reliably discriminate the targeted analytes from background interferences. High resolution and accurate mass (HR/AM) analysis on the recently developed Q-Exactive mass spectrometer can potentially address these issues. This instrument presents a unique configuration: it is constituted of an orbitrap mass analyzer equipped with a quadrupole mass filter as the front-end for precursor ion mass selection. This configuration enables new quantitative methods based on HR/AM measurements, including targeted analysis in MS mode (single ion monitoring) and in MS/MS mode (parallel reaction monitoring). The ability of the quadrupole to select a restricted m/z range allows one to overcome the dynamic range limitations associated with trapping devices, and the MS/MS mode provides an additional stage of selectivity. When applied to targeted protein quantification in urine samples and benchmarked with the reference SRM technique, the quadrupole-orbitrap instrument exhibits similar or better performance in terms of selectivity, dynamic range, and sensitivity. This high performance is further enhanced by leveraging the multiplexing capability of the instrument to design novel acquisition methods and apply them to large targeted proteomic studies for the first time, as demonstrated on 770 tryptic yeast peptides analyzed in one 60-min experiment. The increased quality of quadrupole-orbitrap data has the potential to improve existing protein quantification methods in complex samples and address the pressing demand of systems biology or biomarker evaluation studies.

Dataset Information

Three-minute method for amino acid analysis by UHPLC and high-resolution quadrupole orbitrap mass spectrometry.

Publications

Three-minute method for amino acid analysis by UHPLC and high-resolution quadrupole orbitrap mass spectrometry.

Similar Datasets

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