Quantitative mass spectrometry imaging of amino acids with isomer differentiation in brain tissue via exhaustive liquid microjunction surface sampling-tandem mass tags labeling-ultra performance liquid chromatography-mass spectrometry.

Mass spectrometry imaging (MSI) has been used for localization of various biomolecules in tissues, but it is still challenging to have absolute pixel-to-pixel quantitation of analytes and differentiation of isobaric ions by MSI. In this proof-of-concept study, we present a quantitative MSI method for amino acids with distinguishing their constitutional isomers by exhaustive liquid microjunction surface sampling (LMJSS)–tandem mass tags (TMT) labeling–ultra performance liquid chromatography (UPLC)–MS. TMT6 reagents were used to differentially isotopically label amino acids in extracts from 6 pixels of brain section, resulting in multiplexed analysis of 6 pixels and largely shortening the LC–MSI time. From the results, with TMT labeling, the MS signals of amino acids in brain tissue extract were enhanced by (3–141)-fold. The calibration curves of TMT-labeled amino acids had good linearity in both MS1 and MS2 detection, which is essential for quantitation. A new extraction solvent for LMJSS (10% hexafluoroisopropanol-40% methanol-0.5% acetic acid-water) was developed to improve the extraction efficiencies of polar amino acids from brain tissue. The results showed that the extraction efficiencies of amino acids from different tissue regions were in the range of 75–110% with the new solvent, which made LMJSS an exhaustive sampling. Due to the complete extraction of amino acids from tissue, TMT0-labeled amino acid standards were directly added into the extracts for absolute quantitation. Finally, UPLC–MS was coupled with LMJSS to successfully separate the isobaric labeled amino acids in each pixel, allowing separate imaging of them. The imaging results of amino acid standard pattern demonstrate 500 µm spatial resolution of the MSI method. The brain tissue imaging results showed that the new method enabled quantitative MSI of 11 amino acids including three pairs of isomers, and the quantitation results were highly comparable and correlated with that by traditional bulk extraction–LC–MS method (correlation coefficient = 0.97, the slope of the correlation curve = 0.96).