A chemical derivatization-based pseudotargeted LC-MS/MS method for high coverage determination of dipeptides.

Dipeptides (DPs) have attracted more and more attention in many research fields due to their important biological functions and promising roles as disease biomarkers. However, the determination of DPs in biological samples is very challenging owing to the limited availability of commercial standards, high structure diversity, distinct physical and chemical characteristics, wide concentration range, and the extensive existence of isomers. In this study, a pseudotargeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) method coupled with chemical derivatization for the simultaneous analysis of 400 DPs and their constructing amino acids (AAs) in biospecimens is established. Dansyl chloride (Dns-Cl) chemical derivatization was introduced to provide characteristic MS fragments for annotation and improve the chromatographic separation of DP isomers. A retention time (RT) prediction model was constructed using 83 standards (63 DPs and 20 AAs) based on their quantitative structural retention relationship (QSRR) after the Dns-Cl labeling, which largely facilitated the annotation of the DPs without standards. Finally, we applied this method to investigate the profile change of DPs in a cisplatin-induced acute kidney injury (AKI) rat model. The established workflow provides a platform to profile DPs and expand our understanding of these little-studied metabolites.

High-Coverage Strategy for Multi-Subcellular Metabolome Analysis Using Dansyl-Labeling-Based LC-MS/MS.

Subcellular compartmentalization ensures orderly and efficient intracellular metabolic activities in eukaryotic life. Investigation of the subcellular metabolome could provide in-depth insight into cellular biological activities. However, the sensitive measurement of multi-subcellular metabolic profiles is still a significant challenge. Herein, we present a comprehensive subcellular fractionation, characterization, and metabolome analysis strategy. First, six subcellular fractions including nuclei, mitochondria, lysosomes, peroxisomes, microsomes, and cytoplasm were generated from a single aliquot of liver homogenate. Then, a dansyl-labeling-assisted liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for measuring 151 amino/phenol- or carboxyl-containing metabolites in the subcellular fractions was established and validated. Last, the strategy was applied to a rat model of carbon tetrachloride (CCl4)-induced acute liver injury (ALI). The metabolic profile of individual organelles was compared with that of the liver. Interestingly, many unique changes were observed specifically in organelles, while the liver failed to capture these changes. This result indicates that metabolic investigation at the tissue level might lead to erroneous results due to the leveling effect. Our study demonstrates a feasible approach for the broad-spectrum-targeted metabolic profiling of multi-subcellular fractions, which can be of great use in driving our further understanding of intracellular metabolic activities in various physical and pathological conditions.