title: 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.
authors.affiliation: Laboratory of Protein and Peptide Pharmaceuticals and Laboratory of Proteomics, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.
authors.forename: Tanxi
authors.initials: T
authors.lastname: Cai
authors.affiliation: Laboratory of Protein and Peptide Pharmaceuticals and Laboratory of Proteomics, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China University of Chinese Academy of Sciences, Beijing 100049, China.
authors.forename: Qingbo
authors.initials: Q
authors.lastname: Shu
authors.affiliation: Laboratory of Protein and Peptide Pharmaceuticals and Laboratory of Proteomics, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.
authors.forename: Peibin
authors.initials: P
authors.lastname: Liu
authors.affiliation: Department of Biochemistry and Molecular Biology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430074, China.
authors.forename: Lili
authors.initials: L
authors.lastname: Niu
authors.affiliation: Laboratory of Protein and Peptide Pharmaceuticals and Laboratory of Proteomics, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.
authors.forename: Xiaojing
authors.initials: X
authors.lastname: Guo
authors.affiliation: Laboratory of Protein and Peptide Pharmaceuticals and Laboratory of Proteomics, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.
authors.forename: Xiang
authors.initials: X
authors.lastname: Ding
authors.affiliation: Laboratory of Protein and Peptide Pharmaceuticals and Laboratory of Proteomics, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.
authors.forename: Peng
authors.initials: P
authors.lastname: Xue
authors.affiliation: Laboratory of Protein and Peptide Pharmaceuticals and Laboratory of Proteomics, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.
authors.forename: Zhensheng
authors.initials: Z
authors.lastname: Xie
authors.affiliation: Laboratory of Protein and Peptide Pharmaceuticals and Laboratory of Proteomics, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.
authors.forename: Jifeng
authors.initials: J
authors.lastname: Wang
authors.affiliation: Laboratory of Protein and Peptide Pharmaceuticals and Laboratory of Proteomics, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.
authors.forename: Nali
authors.initials: N
authors.lastname: Zhu
authors.affiliation: Laboratory of Protein and Peptide Pharmaceuticals and Laboratory of Proteomics, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.
authors.forename: Peng
authors.initials: P
authors.lastname: Wu
authors.affiliation: Laboratory of Protein and Peptide Pharmaceuticals and Laboratory of Proteomics, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.
authors.forename: Lili
authors.initials: L
authors.lastname: Niu
authors.affiliation: Laboratory of Protein and Peptide Pharmaceuticals and Laboratory of Proteomics, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China fqyang@ibp.ac.cn.
authors.forename: Fuquan
authors.initials: F
authors.lastname: Yang
mesh_terms: D045744:Cell Line, Tumor; D003978:Diazomethane / Q000378:metabolism; D006801:Humans; D007553:Isotope Labeling; D008246:Lysophospholipids / Q000378:metabolism*; D055432:Metabolomics / Q000379:methods*; D008745:Methylation; D010743:Phospholipids / Q000737:chemistry / Q000378:metabolism*; D013997:Time Factors
chemical_list: D008246:Lysophospholipids; D010743:Phospholipids; D003978:Diazomethane; C032881:lysophosphatidic acid
keywords: chemical synthesis; deuterated diazomethane; hydrogen/deuterium exchange; lipidomics; lysophospholipid; mass spectrometry; trimethylsilyl-diazomethane