Chemical Tagging Assisted Mass Spectrometry Analysis Enables Sensitive Determination of Phosphorylated Compounds in a Single Cell

Abstract

Polar phosphorylated metabolites are involved in a variety of biological processes and play vital roles in energetic metabolism, cofactor regeneration, and nucleic acid synthesis. However, it is often challenging to interrogate polar phosphorylated metabolites and compounds from biological samples. Liquid chromatography–mass spectrometry (LC/MS) now plays a central role in metabolomic studies. However, LC/MS-based approaches have been hampered by the issues of the low ionization efficiencies, low in vivo concentrations, and less chemical stability of polar phosphorylated metabolites. In this work, we synthesized paired reagents of light and heavy isotopomers, 2-(diazomethyl)­phenyl)­(9-methyl-1,3,4,9-tetrahydro-2H-pyrido­[3,4-b]­indol-2-yl)­methanone (DMPI) and d3 -(2-(diazomethyl)­phenyl)­(9-methyl-1,3,4,9-tetrahydro-2H-pyrido­[3,4-b]­indol-2-yl)­methanone (d3 -DMPI). The paired reagents of DMPI and d3 -DMPI carry diazo groups that can efficiently and selectively react with the phosphate group on polar phosphorylated metabolites under mild conditions. As a proof of concept, we found that the transfer of the indole heterocycle group from DMPI/d3 -DMPI to ribonucleotides led to the significant increase of ionization efficiencies of ribonucleotides during LC/MS analysis. The detection sensitivities of these ribonucleotides increased by 25–1137-fold upon DMPI tagging with the limits of detection (LODs) being between 7 and 150 amol. With the developed method, we achieved the determination of all the 12 ribonucleotides from a single mammalian cell and from a single stamen of Arabidopsis thaliana. The method provides a valuable tool to investigate the dynamic changes of polar phosphorylated metabolites in a single cell under particular conditions.

Selective Chemical Labeling Strategy for Oligonucleotides Determination: A First Application to Full-Range Profiling of Transfer RNA Modifications

“…To date, several derivatizing reagents have been reported for quantifying cellular nucleotide pools, including one targeting both the phosphate group and amines, (trimethylsilyl)­diazomethane, and five just targeting the phosphate group, involving 2-(diazomethyl)- N -methyl- N -phenyl-benzamide (2-DMBA), , 8-(diazomethyl)-quinoline (8-DMQ), N , N -dimethyl- p -phenylenediamine (DMPA), 2-(diazomethyl)­phenyl) (9-methyl-1,3,4,9-tetrahydro-2H-pyrido­[3,4- b ]­indol-2-yl) methanone (DMPI), and MTBSTFA . Unfortunately, the introduction of methyl into derivatives by (trimethylsilyl)­diazomethane will confuse the identification of methyl-modified RNA.…”

Advances in the Synthesis and Analysis of Biologically Active Phosphometabolites

“…Tetrabutylammonium acetate has also been used as an ion-pair reagent in an LC-MS method for the fast and robust analysis of negatively charged metabolites, which has been successfully applied to more than 60 common endogenous phosphometabolites and to eight different biological extracts [ 79 ]. In order to overcome challenges in sample preparation and instrumental analysis, such as low concentrations, stability issues, ionization efficiency, recovery, and carry-over effects, chemical derivatization was introduced for analysing specific phosphorylated metabolites from the glycolysis pathway [ 80 ], twelve ribonucleotides in a single cell [ 81 ] as well as 42 phosphomonoesters of S. cerevisiae [ 82 ]. Improvements in phosphometabolites signal intensities, as well as the ratios of the signal to the noise, have also been achieved by column hardware optimization, such as the change from stainless steel to glass and polyethylene materials, or hybrid surface technology [ 83 , 84 ].…”

Hybrid Ionization Source Combining Nanoelectrospray and Dielectric Barrier Discharge Ionization for the Simultaneous Detection of Polar and Nonpolar Compounds in Single Cells

“…Mass spectrometry is a label-free analytical technique with high sensitivity, good selectivity, and fast response. It can simultaneously detect multiple low-molecular-weight metabolites in a cell and has been widely used for single-cell metabolomics. − The ionization method is crucial as it determines the coverage and limit of detection (LOD) of the metabolome. Mass spectrometry-based single-cell metabolomics mainly relies on secondary ion mass spectrometry (SIMS), ,, matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), , and nanoelectrospray ionization mass spectrometry (nanoESI-MS). , SIMS has a high spatial resolution and can be used for subcellular analysis, but many fragments are usually generated during the ionization process.…”