Strategy for Comprehensive Identification of Acylcarnitines Based on Liquid Chromatography–High-Resolution Mass Spectrometry

Abstract

Carnitines play important roles in fatty acid oxidation and branched chain amino acid metabolism. The disturbance of acylcarnitines is associated with occurrence and development of many diseases. Comprehensive acylcarnitine identification can greatly benefit their targeted detection, following disease differential diagnosis and possible mechanism study. In this study, we developed a novel strategy to identify as many acylcarnitines as possible based on liquid chromatography-high-resolution mass spectrometry (LC-HRMS). The layer-layer progressive strategy first integrated the initial full scan MS/data-dependent MS/MS monitoring (ddMS) acquisition and the following parallel reaction monitoring (PRM) to analyze a pooled biological sample. Also 733 possible acylcarnitines were identified containing characteristic high-resolution MS/MS features. Further, accurate mass, retention rules, and HRMS/MS information were used to define subclasses and predict undetected acylcarnitine homologues in each subclass, leading to more acylcarnitines to our newly constructed database. As a result, 758 acylcarnitines were contained in the database, having exact mass, retention time, and MS/MS information, which is the most comprehensive list of acylcarnitines reported to date. Applying this database, 241, 515, and 222 acylcarnitines were rapidly and reliably annotated in human plasma, human urine, and rat liver tissue. This novel strategy enables large-scale identification of acylcarnitines, and a similar method can also be used for identification of other metabolites.

The Structure and Function of Major Plant Metabolite Modifications

“…A widely targeted metabolomics method based on LC-MS has been recently developed for quantifying metabolites in a highthroughput manner, combining the advantages of precise measurement conferred by the targeted profiling and the wide metabolite coverage by the non-targeted analysis (Chen et al, 2013;Yu et al, 2018;Zha et al, 2018), and has been used in major crops including rice (Chen et al, 2014(Chen et al, , 2016, maize (Wen et al, 2014), citrus (Wang et al, 2016(Wang et al, , 2017, and tomato (Zhu et al, 2018). Dai et al (2014) used a combination of insource collision-induced dissociation LC-MS and global non-targeted LC-MS-based metabolomics strategy to study modified metabolites in human urine using characteristic neutral loss fragments that are specific for acetylation, sulfation, glucuronidation, glucosidation, or ribose conjugation.…”

Function, Detection and Alteration of Acylcarnitine Metabolism in Hepatocellular Carcinoma

“…However, the difficulties encountered in the detection of acylcarnitines limit the study of their functions. The challenge for acylcarnitines detection is mostly attributed to the complex components of biological samples and the structural diversity of acylcarnitines caused by various acyl groups [37,59,60]. (1) The composition of biological samples is very complex.…”

“…Due to their excellent separation ability and high sensitivity, dozens of acylcarnitines could be analysed simultaneously [94,95,96,97]. By using highly-selective scanning modes, such as selected reaction monitoring (SRM), multiple reaction monitoring (MRM) and parallel reaction monitoring (PRM), up to hundreds of acylcarnitines could be identified in plasma, urine and tissue samples [37,59,79]. These results provide significant reference value to annotation of acylcarnitines in biological samples.…”

“…This is because the carboxyl compounds are also diverse and lack of sufficient standards. The un-authenticated acylcarnitines in biological samples limited the evaluation of these existing methods [59]. Therefore, the development of new approaches to high efficient and accurately identify acylcarnitines is still in urgent need.…”

Acylcarnitine profiling by low-resolution LC-MS

“…Urine, plasma, blood or dried blood spots are easy to access for screening and are hence the sources of choice for analyzing acylcarnitine deficiencies. A one-dimensional separation of acylcarnitines is commonly done by reversed phase [4–6] or HILIC chromatography [6, 7]. Though a high number of identified molecular species have previously been demonstrated by high-resolution instruments [5–7], quantitative data for comparative studies are only available for a limited number of compounds.…”