A rapid and robust method for amino acid quantification using a simple N-hydroxysuccinimide ester derivatization and liquid chromatography-ion mobility-mass spectrometry.

The vast majority of mass spectrometry (MS)-based metabolomics studies employ reversed-phase liquid chromatography (RPLC) to separate analytes prior to MS detection. Highly polar metabolites, such as amino acids (AAs), are poorly retained by RPLC, making quantitation of these key species challenging across the broad concentration ranges typically observed in biological specimens, such as cell extracts. To improve the detection and quantitation of AAs in microglial cell extracts, the implementation of a 4-dimethylaminobenzoylamido acetic acid N-hydroxysuccinimide ester (DBAA-NHS) derivatization agent was explored for its ability to improve both analyte retention and detection limits in RPLC-MS. In addition to the introduction of the DBAA-NHS labeling reagent, a uniformly (U) 13C-labeled yeast extract was also introduced during the sample preparation workflow as an internal standard (IS) to eliminate artifacts and to enable targeted quantitation of AAs, as well as untargeted amine submetabolome profiling. To improve method sensitivity and selectivity, multiplexed drift-tube ion mobility (IM) was integrated into the LC-MS workflow, facilitating the separation of isomeric metabolites, and improving the structural identification of unknown metabolites. Implementation of the U-13C-labeled yeast extract during the multiplexed LC-IM-MS analysis enabled the quantitation of 19 of the 20 common AAs, supporting a linear dynamic range spanning up to three orders of magnitude in concentration for microglial cell extracts, in addition to reducing the required cell count for reliable quantitation from 10 to 5 million cells per sample.

Design and Implementation of a Dual-Probe Microsampling Apparatus for the Direct Analysis of Adherent Mammalian Cells by Ion Mobility-Mass Spectrometry.

Atmospheric pressure sampling mass spectrometric methods are ideal platforms for rapidly analyzing the metabolomes of biological specimens. Several liquid extraction-based techniques have been developed for increasing metabolome coverage in direct sampling workflows. Here, we report the construction of a dual-probe microsampling device (DPM), based on the design of the liquid microjunction surface sampling probe, for analyzing the metabolome of live microglial cells by drift-tube ion mobility spectrometry (IMS) quadrupole time-of-flight mass spectrometry. Utilizing two distinct solvent systems in parallel is demonstrated to extract a wide structural variety of metabolites and lipids, enabling a more comprehensive analysis of intracellular metabolism. Employing the DPM-IM-MS method to adherent cells yielded the detection of 73 unique lipids and 79 small molecule metabolites from each optimized solvent system probe, respectively. Integration of multiplexed ion mobility scans is also shown to increase extracted analyte signal intensities between 2- and 10-fold compared to traditional single-pulse IMS, enabling the detection of 38 low-intensity features not previously detected by single-pulse DPM-IM-MS. To examine the ability of the DPM system to differentiate between sample treatment groups, microglia were stimulated with the endotoxin lipopolysaccharide (LPS). Several metabolic alterations were detected between sample treatment groups by DPM-IM-MS, many of which were not previously detected with conventional single-probe liquid microjunction surface sampling.