LX Reference Gerl
Contents
Materials and Methods
Chemicals and Lipid Standards
Synthetic lipid standards and a total lipid extract of bovine heart were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL) or Sigma-Aldrich Chemie (Munich, Germany); common chemicals and solvents of ACS or LC–MS grade from Sigma-Aldrich Chemie (Munich, Germany) or Fluka (Buchs SG, Switzerland); methanol (LiChrosolv grade) from Merck (Darmstadt, Germany).
Annotation of Lipid Species
Lipid classes: PE, phosphatidylethanolamines; PE-O, 1-O-alkyl-2-acylglycerophosphoethanolamines; LPE; lyso-phosphatidylethanolamines; PS, phosphatidylserines; PC, phosphatidylcholines; PC-O, 1-O-alkyl-2-acylglycerophosphocholines; LPC, lysophosphatidylcholines; SM, sphingomyelins; PI, phosphatidylinositols; TAG, triacylglycerols; Cer, ceramides; CL, cardiolipins; GlcCer, glucosylceramides.
Individual molecular species were annotated as follows: lipid class no. of carbon atoms in the first fatty acid or fatty alcohol moiety:no. of double bonds in the first fatty acid or fatty alcohol moiety/no. of carbon atoms in the second fatty acid moiety:no. of double bonds in the second fatty acid moiety. For example, PC 18:0/18:1 stands for a phosphatidylcholine comprising the moieties of stearic (18:0) and oleic (18:1) fatty acids. If the exact composition of fatty acid or fatty alcohol moieties was unknown, the species were annotated as lipid class no. of carbon atoms in both moieties:no. of double bonds in both moieties. In this way, PC 36:1 stands for the PC species having 36 carbon atoms and one double bond in both fatty acid moieties.
Total lipid extract from rat retina was prepared as described(35) with minor modifications. Briefly, a sample of rat retina tissue (wet weight 7 mg) was homogenized using a Dounce tissue grinder (Sigma-Aldrich Chemie, Munich) and extracted with 700 μL of methyl-tert-butyl ether (MTBE)/methanol 10:3 (v/v). Synthetic standards of PC 17:0/17:0; PE 17:0/17:0; PS 12:0/12:0, and PI 8:0/8:0 were spiked into the MTBE/MeOH mixture prior to lipid extraction in the concentration of 10 μM each and the mixture was incubated at 4 °C for 1 h on a shaking platform. To initiate phase separation, 135 μL of water were added and the mixture was shaken for another 15 min. Phase separation was completed upon centrifuging for another 5 min in a benchtop centrifuge MiniSpin (Eppendorf, Hamburg) at 13 400 rpm. The upper organic phase was collected and stored at −25 °C under nitrogen.
Samples for Mass Spectrometric Analysis
Retina total lipid extracts or lipid standards were diluted with a mixture of isopropanol/methanol/chloroform 4:2:1 (v/v/v) containing 7.5 mM ammonium formate or ammonium acetate, as indicated separately for each experiment. Also, where specified, we used a mixture of methanol/chloroform 5:1 (v/v) containing 0.1% (v/v) triethylamine. Prior to the analysis, samples were loaded into a 96-well plate (Eppendorf, Hamburg), sealed with aluminum foil, and centrifuged for 5 min at 4 000 rpm on a Multifuge 3S-R centrifuge from Heraeus DJB Labcare Ltd. (Newport Pagnell, U.K.). The retina extract was diluted 10 times prior to the analysis. Final concentrations of synthetic standards were selected individually for each experiment. Mass spectrometric analses were performed on the LTQ Orbitrap XL (further termed as XL) and LTQ Orbitrap Velos (further termed as Velos) instruments (Thermo Fisher Scientific, Bremen), both equipped with a robotic nanoflow ion source TriVersa (Advion BioSciences, Ithaca NY) using chips with spraying nozzles with a diameter of 4.1 μm. The ion source was controlled by Chipsoft 6.4 software. The ionization voltage and gas backpressure were set to 1.25 kV and 0.95 psi in the positive and 0.7 kV and 1.06 psi in negative ion modes, respectively. Under these settings, 10 μL of the analyte was electrosprayed for more than 30 min.
The temperatures of the ion transfer capillary were 125 and 200 °C for the XL and Velos, respectively; the tube voltages were 90 V (MS+) or −150 V (MS−) for both machines; and the s-lens level was 58% for the Velos. Isolation of Lipid Precursor Ions on XL and Velos Machines The abundance of isolated precursors was monitored by the method of total ion mapping. To this end, MS/MS spectra were acquired with the ion trap (IT) or (where specified) with the Orbitrap (FT) analyzer, while the target precursor m/z was changed with step increments of 0.1 Th. Precursor isolation width (1.0 Th); maximum injection time (100 ms), automated gain control (AGC) (5 000 ions), and normalized collision energy (nCE) (1%) were fixed, while the target precursor m/z was altered in 0.1 Th step increments.
Optimal offset values for species of different lipid classes were determined by analyzing in replicate in the negative ion mode a mixture of synthetic lipid standards LPC 12:0, PC 12:0/12:0, PC 14:0/14:0, PC 22:0/22:0, PC 24:0/24:0, and Cer d18:1/17:0; CL 14:0/14:0/14:0/14:0, GlcCer d18:1/17:0, PG 17:0/17:0, SM d18:1/17:0, and PC 17:0/17:0, each at a concentration of 2.5 μM. Total ion maps were acquired under the following settings: nCE was 1%; isolation width was 1.5 Th; ITmax was 250 ms; target value for AGC was 5 000; and target mass resolution at m/z 400 (Rm/z400) was 7 500 (full width at half maximum, FWHM).
Lipid fragmentation in collision induced dissociation (CID), pulsed Q collision induced dissociation (PQD), and HCD modes was compared by acquiring collision energy profiles for the synthetic lipid standard PE 18:1/18:2. The analyte with a concentration of 0.5 μM was infused into a mass spectrometer in the negative ion mode, and the abundances of precursor and fragment ions were plotted against the normalized collision energy. MS/MS spectra were acquired under the following common settings: isolation width was 1.5 Th; ITmax was 100 ms; AGC value was set to 25 000; target mass resolution Rm/z400 was 30 000; for each spectrum, 3 scans were averaged within the total acquisition time of 1.19 s.
Identification and Quantification of Molecular Species in Total Lipid Extracts
Centroided HCD FT MS/MS spectra were acquired in data-dependent mode as described.(19) Each data-dependent acquisition (DDA) cycle consisted of one FT MS survey spectrum acquired at the target resolution Rm/z400 of 100 000, followed by the acquisition of five HCD FT MS/MS spectra at the resolution Rm/z400 of 30 000. The prescan option was disabled. Precursor ions were subjected to MS/MS if their m/z matched the masses in the inclusion list with the accuracy of ±5 ppm. In MS/MS experiments, precursor ions were isolated at the linear ion trap with the isolation width of 1.6 Th, and the ITmax was set to 4 s. Exclusion time was set to 20 min. On average, a DDA experiment was completed in 40 min such that each precursor was fragmented twice. In HCD mode, nCE was 45%; fragments (m/z ≥ 100) were detected by the Orbitrap analyzer at the resolution Rm/z400 of 30 000. The maximum scan time for acquiring one HCD FT MS/MS spectrum was 5.4 s. The lock mass option was enabled and abundant background anion of octadecyl-(di-tert-butyl-hydroxyphenyl)propionate ([M – H]−) with m/z 529.46262 was used as a reference peak. Target AGC values were set to 1 × 106 and 2.5 × 104 for FT MS and FT MS/MS modes, respectively. In FT MS, ITmax was 100 ms and 3 scans were averaged for each spectrum. DDA experiments were repeated four times for each sample and twice for the blank.
Lipid species were identified by LipidXplorer software as described in the Supporting Information, part 1. Lipid species were quantified by comparing the abundances of precursor peaks in high-resolution MS spectra and acyl anions peaks in MS/MS spectra with corresponding peaks of precursors and fragments of spiked internal standards.(19, 20) For quantifying rat retina lipids, abundances of monoisotopic peaks were adjusted according to isotopic profiles calculated from elemental compositions of corresponding molecular ions to compensate the large difference in the length of fatty acid moieties.
