Development of Tandem Mass Tag Labeling Method for Lipid Molecules Containing Carboxy and Phosphate Groups, and Their Stability in Human Serum

Abstract

In clinical lipidomics, it is a challenge to measure a large number of samples and to reproduce the quantitative results. We expanded the range of application of the tandem mass tag (TMT) method, which is widely used in proteomics, to lipidomic fields. There are various types of lipid molecule, for example, eicosanoids have a carboxyl group and phosphatidic acid has a phosphate group. We modified these functional groups simultaneously with TMT. This approach allows for a single analysis by mixing six samples and using one of the six samples as a bridging sample; the quantitative data can be easily normalized even if the number of measurements increases. To accommodate a large number of samples, we utilize a pooled serum sample of 300 individuals as a bridging sample. The stability of these lipid molecules in serum was examined as an analytical validation for the simultaneous TMT labeling. It was found that the stability of these lipid molecules in serum differs greatly depending on the lipid species. These findings reaffirmed the importance of proper sample preparation and storage to obtain reliable data. The TMT labeling method is expected to be a useful method for lipidomics with high-throughput and reliable reproducibility.

The Second Asia-Oceania Human Proteome Organization (AOHUPO) Online Education Series on the Renaissance of Clinical Proteomics: Biomarkers, Imaging and Therapeutics

“…Interestingly, he found no overlap between PEA and LC-MS datasets acquired from plasma samples. He next presented an in-house–developed tandem mass tags-lipidomics technique that can label diverse classes of lipids ( 4 , 5 ). These techniques were then combined to investigate the plasma samples from elderly Japanese with different degrees of Alzheimer’s disease (AD).…”

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

“…Although high-throughput quantitative analysis strategies have been extensively implemented in proteomic and glycomic studies, limited work has been performed on FA analysis in a high-throughput manner. For example, AminoxyTMT, a chemical tag for carbonyl-containing biomolecules, was used for multiplex FA analysis. , Accurate quantitation was achieved by LC–MS analysis in the positive mode, while the structural elucidation of unsaturated FAs remains challenging.…”

“…For example, AminoxyTMT, a chemical tag for carbonyl-containing biomolecules, was used for multiplex FA analysis. 21,22 Accurate quantitation was achieved by LC−MS analysis in the positive mode, while the structural elucidation of unsaturated FAs remains challenging. Recently, our group developed 4-plex SUGAR and 3-plex mdSUGAR tags for quantitative glycomics.…”

Lipid Isobaric Mass Tagging for Enhanced Relative Quantification of Unsaturated sn-Positional Isomers

“…Isobaric tag labeling, such as iTRAQ (isobaric tag for relative and absolute quantitation) and TMT (tandem mass tag) labeling, becomes a robust quantification method in proteomics, which offers ideal reproducibility, precision, and the ability to multiplex samples without the use of ISs. − These tags contain a reactive group for selective protein and peptide modification, a mass balancer, and a mass reporter. Each isobaric reagent comprises a distinctive set of light and heavy isotopes distributed between the balancer and reporter regions.…”

“…In contrast to proteins, lipids exhibit structural diversity, with numerous isomeric forms, and lack common functional groups suitable for tag labeling. Current isobaric tags are only applicable to quantify lipids with amino, carboxyl, and phosphate groups and are not suitable for lipid isomer quantification. − Consequently, there is an urgent need for the development of lipid isobaric tags to facilitate the quantification of lipids across various categories at the isomer level. Knowing the prevalence of unsaturated lipids within the overall lipid categories, our previous work reported a lipid isobaric tag labeling strategy that functionalizes lipid CC bonds via N -H aziridination followed by the introduction of isobaric tags (TMT) through N -hydroxysuccinimide (NHS) chemistry .…”