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.

Fig. 1.

Electrospray ionization mass spectra in the negative-ionization mode of crude lipid extracts of Sinorhizobium meliloti P110 grown in MOPS medium. A: Full-scan mode; insert shows an expanded view of m/z 700–760. B: Precursor ion scan of m/z 153. C: Precursor ion scan of m/z 225. I.S., internal standard; PG, phosphatidylglycerol; SL, sulfoquinovosyldiacylglycerol.

Fig. 2.

Electrospray ionization mass spectra in the positive-ion mode of crude lipid extracts of S. meliloti P110. A: Full-scan mode. B: Full-scan mode, 10 mM LiCl added. C: Neutral-loss scan of 147 mass units. D: Neutral-loss scan of 161 mass units. E: Neutral-loss scan of 175 mass units. F: Neutral-loss scan of 189 mass units. DMPE, di-methylphosphatidylethanolamine; MMPE, monomethylphosphatidylethanolamine; PC, phosphatidylcholine; PE, phosphatidylethanolamine.

Fig. 3.

Positive-ion electrospray ionization mass spectra of crude lipid extracts of S. meliloti P110 grown under P_i-starvation conditions. A: Full-scan mode spectrum with no LiCl added. B: Full-scan mode spectrum with 10 mM LiCl added. C: Neutral-loss scan of 74 mass units selective for TMHSs. D: Precursor ion scan of m/z 115 selective for OLs. OL, ornithine lipid; PE, phosphatidylethanolamine; PC, phosphatidylcholine; TMHS, 1,2-diacylglyceryl-3-O-4′-(N,N,N-trimethyl)-homoserine lipid.

Fig. 4.

Principal component analysis of relative percentage composition in lipids of S. meliloti P110 under normal growth conditions (A) and under P_i-starvation conditions (B). Lipid classes are represented by numbers: 1, PGs; 2, PEs; 3, MMPEs; 4, DMPEs; 5, PCs; 6, SLs; 7, TMHs; 8, OLs.

Fig. 5.

Positive electrospray ionization mass spectra in the full-scan mode of crude lipid extracts of wild type (A) and phoB knockout mutant (B) under P_i-starvation conditions.