Lipids Structural Characterization of a Single Horse Embryo by Mass Spectrometry.

Abstract

Matrix-assisted laser desorption-ionization mass spectrometry (MALDI-MS) allows not only the fast and efficient detection of various lipids classes, but also the study of their structure by fragmentation (MS/MS) experiments (Ferreira et al., 2010. J Lipid Res, 51: 1218–1227). The aim of this study was to characterize the lipid classes present in one single equine embryo by MALDI-MS, and to study some lipid structures by collision induced dissociation (CID) experiments. One equine embryo recovered by uterine wash on day 8 (D8) post ovulation, was placed in 50/50 (v/v) methanol/phosphate buffer solution and transported at 4ºC to the laboratory. MALDI-MS spectra were acquired in the positive (using 2,5-dihydroxybenzoic acid as MALDI matrix) and negative (using -aminoacridine hemihydrate as MALDI matrix) ion modes in a MALDI Synapt HDMS mass spectrometer (Waters, Manchester, UK) at the m/z 400–1000 range. Spectra processing was performed using the MassLynx 4.0 software (Waters, Manchester, UK). Besides obtaining the lipid profile at both ion modes, some ions were isolated in the positive and in the negative ion mode for MS/MS experiments. In the lipid profile in the positive ion mode, mainly phosphatidylcholines (PC), phosphatidylethanolamines (PE), sphingomielyns (SM), and triacylglyeceols (TAG) species were observed, while in the negative ion mode, PE and phospathidylinositols (PI) were detected. MS/MS spectrum in the positive mode of m/z 760.6 (attributed as PC34:1) depicted characteristic PC fragments of m/z 184.1 (choline polar head), and the neutral loss (NL) of 183 (phosphorylcholine). For the ion of m/z 766.6 (attributed as PE 38:5), we observe the NL of 140, characteristic of PE. For the ion of m/z 808.7 (attributed as PC 38:5), besides the fragment at m/z 184.1 at the NL of 183, it was possible to observe the loss of trimethylamine (ion at m/z 749.6), and the cyclophosphane (ion at m/z 147.0). Finally, for the negative ion mode, we isolated and fragmented the ion at m/z 863.6, which was attributed as PI 36:1 due to the presence of various characteristic ions reported for this lipid species: At m/z 153 (glycerol phosphate -H2O-H), 223 (phospho inositol-2H2O-H), 241 (phospho inositol-H2O-H), 281 (oleic acid), and 581.3 (lysophosphoinositol-H2O-H). We conclude that MALDI-MS allows the detection of PC, SM, PE, PI and TAG lipid species, as well as fast and confident characterization of its lipid structure from one single equine embryo. These results may contribute to a better knowledge of equine embryo metabolism and to cryopreservation studies.Support from São Paulo Research Foundation (FAPESP) are gratefully acknowledged.