Quantitative Analysis and Structural Elucidation of Fatty Acids by Isobaric Multiplex Labeling Reagents for Carbonyl-Containing Compound (SUGAR) Tags and m-CPBA Epoxidation

Abstract

In this study, a novel analytical method was developed to investigate fatty acids (FAs) for relative quantification, carbon–carbon double-bond localization, and cis-/trans-geometry differentiation by isobaric multiplex labeling reagents for carbonyl-containing compound (SUGAR) tag conjugation and meta-chloroperoxybenzoic acid (m-CPBA) epoxidation. FAs are essential components of cells and have diverse functions in energy storage and as complex lipid constituents. It has been reported that FAs play different roles in various biological processes such as the functional development of the brain. The comprehensive characterization and quantification of FAs are crucial to further elucidate their biological roles. However, it is challenging to perform relative quantification and structural elucidation of FAs using integrated mass spectrometry (MS)-based methods. Recently, our group developed isobaric multiplex SUGAR tags for quantitative glycomics. Besides aldehyde/ketone groups on glycans, hydrazide groups also possess reactivity toward carboxylic acids on FAs. In this study, we extended SUGAR tag labeling with FAs for the quantitative analysis by liquid chromatography (LC)-MS/MS in the positive ion mode and applied this strategy for the comparative analysis of FAs hydrolyzed from oil samples. In addition, to comprehensively elucidate the structures of unsaturated FAs, epoxidation by m-CPBA was performed before SUGAR tag labeling to enable carbon–carbon double-bond localization. Moreover, the cis- and trans-geometries of carbon–carbon double bonds in multiple pairs of monounsaturated FAs could also be differentiated in higher-energy collisional dissociation (HCD)-MS/MS. This study developed a high-throughput comprehensive FA analysis platform, which could be widely applied and utilized in biological and clinical studies.

High-Throughput Quantitative Glycomics Enabled by 12-plex Isobaric Multiplex Labeling Reagents for Carbonyl-Containing Compound (SUGAR) Tags

“…The structure of the SUGAR tag consists of three moieties: a reporter group, a balancer group, and a reactive group that enables selective modification of the free reducing end of the released glycans. The reporter group utilizes N , N -dimethyl leucine (DiLeu) benefiting from its excellent fragmentation performance in glycomics, lipidomics, proteomics, and glycopeptidomics. , The balancer group consists of glycine as a linker while a hydrazide group is used as the reactive group to incorporate the SUGAR tag into the released glycans through reductive amination , (Figure ).…”

“…The reporter group utilizes N,N-dimethyl leucine (DiLeu) benefiting from its excellent fragmentation performance in glycomics, lipidomics, proteomics, and glycopeptidomics. 17,18 The balancer group consists of glycine as a linker while a hydrazide group is used as the reactive group to incorporate the SUGAR tag into the released glycans through reductive amination 19,20 (Figure 1).…”

Identification of Carbon-Carbon Double Bond Stereochemistry in Unsaturated Fatty Acids by Charge-Remote Fragmentation of Fixed-Charge Derivatives

“…While instances of co-elution and non-specific CRF were identified, the structural assignments could be confirmed by independent analysis using LC-OzID protocols. In contrast to previously reported strategies, the herein described method utilizes a single derivatization procedure, with stereo-isomer-specific product ions formed in both beam- and trap-type activations, suggesting ready implementation of a range of instruments currently used for lipidomics. Similarly, given the utilization of conventional LC and MS equipment and compatibility with standard data analysis protocols for LC-MRM workflows, this approach will prove advantageous for FA identification in many instances.…”

“…Recently, dualderivatization strategies, that include epoxidation of carboncarbon double bonds, have been shown to effectively elucidate DB position, while differences in product ion abundances reveal DB stereochemistry under controlled ion activation conditions. 35 Using the amide-coupled, fixed-charge derivatives, NMPA, and AMPP (Figure S1), we report the LC-MS/MS separation and unambiguous identification of a suite of monounsaturated fatty acid isomers differing in double bond position and cis/ trans-geometry. Assisted by the robust amide linker and the fixed positive charge, CRF is more competitive with chargedriven processes in these derivatized FAs.…”

“…In addition to CID, fixed charge-derivatives have also been used in conjunction with other ion activation modalities including ozone-induced dissociation, , Paternò-Büchi derivatization, and photodissociation to provide assignment of site(s) of unsaturation and chain branching, and thus, differentiation of isomeric fatty acids. Recently, dual-derivatization strategies, that include epoxidation of carbon-carbon double bonds, have been shown to effectively elucidate DB position, while differences in product ion abundances reveal DB stereochemistry under controlled ion activation conditions …”