A shotgun lipidomics approach in Sinorhizobium meliloti as a tool in functional genomics

Abstract

A shotgun lipidomics approach that allowed the analysis of eight lipid classes directly from crude extracts of the soil bacterium Sinorhizobium meliloti is presented. New MS-MS transitions are reported for the analysis of monomethylphosphatidylethanolamines, dimethylphosphatidylethanolamines, and three bacterial non-phosphorus-containing lipid classes [sulfoquinovosyldiacylglycerols, ornithines, and diacylglyceryl-(N,N,N-trimethyl)-homoserines]. Unique MS-MS transitions allowed the analysis of isomeric species from various lipid classes without chromatography. Analyses required small sample amounts and minimal preparation; thus, this methodology has excellent potential to be used as a screening tool for the analysis of large numbers of samples in functional genomics studies. FA distributions within lipid classes of S. meliloti are described for the first time, and the relative positions of fatty acyl substituents (sn-1, sn-2) in phospholipids are presented. FA distributions in diacylglyceryl-(N, N,N-trimethyl)-homoserines were identical to those of phospholipids, indicating a common biosynthetic origin for these lipids. The method was applied to the analysis of mutants deficient in the PhoB regulator protein. Increased lipid cyclopropanation was observed in PhoB-deficient mutants under P i starvation.-Basconcillo, L. S., R. Zaheer, T. M. Finan, and B. E. McCarry. A shotgun lipidomics approach in Sinorhizobium meliloti as a tool in functional genomics.

Identification of Phosphomethylethanolamine N-Methyltransferase from Arabidopsis and Its Role in Choline and Phospholipid Metabolism

“…PtdMEA components are frequently near or below the level of detection, and PtdDEA lipid molecular species were not statistically different from the detection limits. Although absolute quantitative estimates for components in profiles can be difficult to make (30,36), the peak heights associated with PtdMEA are consistent with this species being a minor component of total phospholipids (Ͻ2%). However, the 34:3-PtdMEA was significantly different between the genotypes, being, on average, 2.1-fold more abundant among phospholipids of atpmeamt plants (Figs.…”

“…Across neutral loss profiles, for each additional methyl group added to a given lipid with a specific fatty acid composition, the m/z increases by a factor of 14. As such, neutral losses of 147, 161, 175, and 189 correspond to the lithiated polar headgroups of PtdEA, PtdMEA, PtdDEA, and PtdCho, respectively (30). The ESI-MS/MS profiles for phospholipids extracted from leaves of wild-type Arabidopsis plants are on the same scale to show the relative abundance of each Ptd-base species in a given tissue extract.…”

“…Fig. 6 shows representative phospholipid profiles in positive scan mode, with neutral losses corresponding to the mass of the polar headgroup (30,36). Across neutral loss profiles, for each additional methyl group added to a given lipid with a specific fatty acid composition, the m/z increases by a factor of 14.…”

“…Lipidomics analysis was conducted on the chloroform phase using infusion electrospray ionization-mass spectrometry (ESI-MS) in the positive ion mode by the McMaster Regional Centre for Mass Spectrometry according to the method of Basconcillo et al (30). To the lipid (chloroform) phase was added 200 l of methanol/chloroform (1:1, v/v).…”

“…Typically, 100 spectra were collected and averaged to afford a single spectrum for each lipid analysis. MS/MS analyses were used to determine the identity of the fatty acyl groups in the lipids (30).…”