Multiplexed analysis of amino acids in mice brain microdialysis samples using isobaric labeling and liquid chromatography-high resolution tandem mass spectrometry

Juho Heininen,Ulrika Julku,Timo Myöhänen,Tapio Kotiaho,Risto Kostiainen

doi:10.1016/j.chroma.2021.462537

Abstract

We developed a new multiplexed reversed phase liquid chromatography-high resolution tandem mass spectrometric (LC-MS/MS) method. The method is based on isobaric labeling with a tandem mass tag (TMT10-plex) and stable isotope-labeled internal standards, and was used to analyze amino acids in mouse brain microdialysis samples. The TMT10-plex labeling of amino acids allowed analysis of ten samples in one LC-MS/MS run, significantly increasing the sample throughput. The method provides good chromatographic performance (peak half-width between 0.04–0.12 min), allowing separation of all TMT-labeled amino acids with acceptable resolution and high sensitivity (limits of detection typically around 10 nM). The use of stable isotope-labeled internal standards, together with TMT10-plex labeling, ensured good repeatability (relative standard deviation ≤ 12.1 %) and linearity (correlation coefficient > 0.994), indicating good quantitative performance of the multiplexed method. The method was applied to study the effect of d-amphetamine microdialysis perfusion on amino acid concentrations in the mouse brain. All amino acids were reliably detected and quantified, indicating that the method is sensitive enough to detect low concentrations of amino acids in brain microdialysis samples.

Highlights

Mass spectrometry combined with chromatographic methods has largely been applied to quantitative bioanalysis
Internal standard method, which often uses stable isotope dilution methodology, provides high reliability for quantitative analysis [1]. This is because the method can compensate for the possible variabilities in sample preparation or suppression in ionization, especially when electrospray ionization is used in liquid chromatography-mass spectrometry (LC-MS)
The use of higher concentrations of tandem mass tag (TMT) result in increased costs and in increased concentrations of TMT byproducts, which may disturb the analysis of amino acids. 20-fold excess of TMT labeling reagent has been found to be suitable in the earlier NHS-based labeling methods [33,34]

Summary

Introduction

Mass spectrometry combined with chromatographic methods has largely been applied to quantitative bioanalysis. Quantitative methods can be based on the use of external or internal standards. Internal standard method, which often uses stable isotope dilution methodology, provides high reliability for quantitative analysis [1]. This is because the method can compensate for the possible variabilities in sample preparation or suppression in ionization, especially when electrospray ionization is used in liquid chromatography-mass spectrometry (LC-MS). This is important for the quantitative analysis of complex biological samples. Multiplexing can be achieved with MS resolvable mass difference labeling or tandem mass spectrometry (MS/MS or MS2)

Methods

Results

Conclusion