Characterization and relative quantification of phospholipids based on methylation and stable isotopic labeling

Abstract

Phospholipids (PLs), one of the lipid categories, are not only the primary building blocks of cellular membranes, but also can be split to produce products that function as second messengers in signal transduction and play a pivotal role in numerous cellular processes, including cell growth, survival, and motility. Here, we present an integrated novel method that combines a fast and robust TMS-diazomethane-based phosphate derivatization and isotopic labeling strategy, which enables simultaneous profiling and relative quantification of PLs from biological samples. Our results showed that phosphate methylation allows fast and sensitive identification of the six major PL classes, including their lysophospholipid counterparts, under positive ionization mode. The isotopic labeling of endogenous PLs was achieved by deuterated diazomethane, which was generated through acid-catalyzed hydrogen/deuterium (H/D) exchange and methanolysis of TMS-diazomethane during the process of phosphate derivatization. The measured H/D ratios of unlabeled and labeled PLs, which were mixed in known proportions, indicated that the isotopic labeling strategy is capable of providing relative quantitation with adequate accuracy, reproducibility, and a coefficient of variation of 9.1%, on average. This novel method offers unique advantages over existing approaches and presents a powerful tool for research of PL metabolism and signaling. Phospholipids (PLs), one of the lipid categories, are not only the primary building blocks of cellular membranes, but also can be split to produce products that function as second messengers in signal transduction and play a pivotal role in numerous cellular processes, including cell growth, survival, and motility. Here, we present an integrated novel method that combines a fast and robust TMS-diazomethane-based phosphate derivatization and isotopic labeling strategy, which enables simultaneous profiling and relative quantification of PLs from biological samples. Our results showed that phosphate methylation allows fast and sensitive identification of the six major PL classes, including their lysophospholipid counterparts, under positive ionization mode. The isotopic labeling of endogenous PLs was achieved by deuterated diazomethane, which was generated through acid-catalyzed hydrogen/deuterium (H/D) exchange and methanolysis of TMS-diazomethane during the process of phosphate derivatization. The measured H/D ratios of unlabeled and labeled PLs, which were mixed in known proportions, indicated that the isotopic labeling strategy is capable of providing relative quantitation with adequate accuracy, reproducibility, and a coefficient of variation of 9.1%, on average. This novel method offers unique advantages over existing approaches and presents a powerful tool for research of PL metabolism and signaling. Phospholipids (PLs) have diverse and critical roles in cellular metabolism and function, such as cell growth, survival, and motility (1Escribá P.V. González-Ros J.M. Goñi F.M. Kinnunen P.K. Vigh L. Sánchez-Magraner L. Fernández A.M. Busquets X. Horváth I. Barceló-Coblijn G. Membranes: a meeting point for lipids, proteins and therapies.J. Cell. Mol. Med. 2008; 12: 829-875Crossref PubMed Scopus (279) Google Scholar). First, PLs are the primary building blocks of cellular membranes, which hold the living matter within each cell and also give definition, shape, and protection to many of the organelles within cells. Second, some types of PLs can be split to produce products that function as second messengers in signal transduction (2Nishizuka Y. Intracellular signaling by hydrolysis of phospholipids and activation of protein kinase C.Science. 1992; 258: 607-614Crossref PubMed Scopus (4224) Google Scholar). Moreover, aberrant PL metabolism has been implicated in numerous human diseases, such as cancer, neurological disorders, diabetes, and others (3Dória M.L. Cotrim Z. Macedo B. Simoes C. Domingues P. Helguero L. Domingues M.R. Lipi