Total Lipid Profiling LTQ-FT

From LipidomicsWiki

Chromatographic methods
High-performance liquid chromatography
The Accela HPLC system was equipped with a reversed-phase C18 column (reversed-phase C18; 100 x 1 mm i.d., 1.9 µm particle size), both from Thermo Fisher Scientific, San Jose, CA, USA. Mobile phase A was 10 mM ammonium acetate containing 0.1% formic acid. Mobile phase B was acetonitrile/2-Propanol 5:2 (v/v) containing 10 mM ammonium acetate and 0.1% formic acid. The binary gradient started with 35 to 70% B for 4 min, then was raised up to 100% B in another 16 min and further held for 10 min. The flow rate was 250 µl/min, the oven temperature was 50°C and tray temperature 10°C. For analysis 5 µL sample were injected. After each run the column was flushed 5 min with 35% B before the next run was started.

High-performance liquid chromatography for 2D chromatographic separation
HILIC-HPLC was carried out in an Agilent 1100 HPLC system (Waldbronn, Germany) consisting of a degasser, a binary pump, a thermostated autosampler and a thermostated column compartment equipped with a semi-preparative column, filled with Nucleosil 100-5 OH, 250 x 10 mm i.d., 5.0 µm particle size (Macherey-Nagel, Düren, Germany). Mobile phase A was cyclohexane and mobile phase B was 2-Propanol/deionized water/acetic acid/28% of ammonia 86:13:1:0.12 (v/v). The HPLC flow rate was 1000 µL/min at an isocratic composition of 10% A and 90% B, at 35°C oven and 5°C tray temperature. The injection volume was 100 µL. For online monitoring of lipid fractionation the HPLC system was coupled in positive ESI-mode to a 4000 quadrupole linear ion trap mass spectrometer (Q-Trap) (Applied Biosystem/MDS Sciex, Concord, ON, Canada) with a split of 1:21. Two fractions were manually collected and each subjected to desalting and concentration. For this, 10 mL deionized water was added to each fraction and the organic phase collected. After re-extraction of the aqueous phase with chloroform/methanol 1:1 (v/v), combined organic phases were dried in a SpeedVac and taken up again in 100 µL chloroform/methanol 1:1 (v/v) for further analysis by reversed-phase-HPLC as described before.

Mass spectrometry
LTQ-FT mass spectrometry
A 7.0 Tesla LTQ-FT hybrid linear ion trap Fourier transform ion cyclotron resonance mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) equipped with an electrospray ion source was used. The instrument was operated in preview mode for parallel MS/MS spectra in the linear ion trap, while running the ion cyclotron in full scan mode at 200,000 resolution (m/z 400) from m/z 400 to 1050 in positive and from m/z 350 to1050 in negative ESI-mode. Helium was used as gas for linear ion trap collision-induced dissociation (CID) spectra. From the LTQ-FT preview scan the 4 most abundant ions were selected in data dependent acquisition (DDA), fragmented in the linear ion trap analyzer and ejected at nominal mass resolution. The following parameters were used for positive and negative ESI-MS/MS experiments: Normalized collision energy was 35%, the repeat count was 2 and the exclusion duration 60 s. The activation Q was at 0.2 and the isolation width 2. For positive ESI spray voltage was set to 5 kV and the tube lens offset was at 120 V. For negative ESI spray voltage was - 4.8 kV and the tube lens offset was - 87 V. The sheath gas flow was set to 50 arbitrary units, auxiliary gas flow to 20 arbitrary units, sweep gas flow to 2 arbitrary units and the capillary temperature to 250°C.

Data analysis
Identification of lipids was performed by LDA, a platform independent Java application as detailed elsewhere (7). Briefly, the algorithm identifies peaks in a three dimensional (3D) distribution of retention time, m/z and intensity, integrates them, and identifies lipids by matching exact mass and isotopic distribution to theoretical values from a database.