Sphingolipid profiling - LC-MS/MS - Scherer et al.
From LipidomicsWiki
Contents |
Sample preparation
- sphingolipids were extracted with butanol
- internal standards were added prior lipid extraction
- supernatant was transferred to a glass vial and injected
Material | Material used | Internal Standard(s) | Internal Standard(s) added |
---|---|---|---|
Cell homogenate | 100µg | SPH d17:1 | 20 ng |
| | SPC d17:1 | 2 ng |
| | GluCer 12:0 | 20 ng |
| | LacCer 12:0 | 20 ng |
| | Cer1P 12:0 | 20 ng |
Instrumentation and method
Pump
- Type: binary and isocratic pump (Agilent 1200)
- Mode: gradient elution
- Solvent(s): Solvent A water containing 0.2% formic acid and 200 mM ammonium formate ; Solvent B acetonitrile containing 0.2% formic acid
- column flow was directed only from 1.0 to 3.0min into the mass spectrometer; otherwise methanol with a flow rate of 250µL/min was delivered
- Gradient:
Time [min] | Flow [ml/min] | % Solvent A | % Solvent B |
---|---|---|---|
0 | 0.8 | 0 | 100 |
0.1 | 0.8 | 0 | 100 |
0.11 | 0.8 | 10 | 90 |
2.5 | 0.8 | 50 | 50 |
3.5 | 0.8 | 50 | 50 |
3.51 | 0.8 | 0 | 100 |
4.5 | 0.8 | 0 | 100 |
Autosampler
- Type: CTC Pal
- Injection volume: 2µl
- Wash solvent 1: Isopropanol/MeOH (1/1; v/v), Wash solvent 2: MeOH
Column
- Type: Interchim HILIC column (Interchim, Montlucan, France)
- length: 50 x 2 mm i.d., 0.5 µm pre-column filter
- Particle size: 1.8 µm
- Column temperature: 50°C
Mass spectrometer
- Type: Triple quadrupole (Applied Biosystems, 4000 QTRAP)
- Ionization mode: ESI positive
- Ionization voltage: 5500V
- Source temperature: 400°C
- Collision gas: Nitrogen
- Collision gas pressure: medium
- MS/MS-conditions:
- precursor ion scan of m/z 264 for HexCer, LacCer, Cer1P d18:1 species screening
- neutral loss scan of m/z 98 for Cer1P d18:0 species screening
- multiple reaction monitoring table of species observed frequently
Analyte | Precursor [m/z] | Product [m/z] | Collision energy [eV] |
---|---|---|---|
SPH d18:1 | 300.3 | 282.2 | 17 |
| 300.3 | 252.2 | 25 |
SPH d18:0 | 302.3 | 284.2 | 21 |
| 302.3 | 254.2 | 29 |
SPH t18:0 | 318.3 | 282.2 | 23 |
Dimethyl-SPH d18:1 | 328.3 | 280.3 | 29 |
Trimethyl-SPH d18:1 | 342.3 | 60.1 | 49 |
SPC d18:1 | 465.3 | 184 | 31 |
HexCer d18:1 | [M+H]+ | 264.3 | 55 |
LacCer d18:1 | [M+H]+ | 264.3 | 65 |
Cer1P d18:1 | [M+H]+ | 264.3 | 39 |
Cer1P d18:0 | [M+H]+ neutral loss scan of 98 | 29 |
Data analysis and quantification
Software
- Analyst 1.4.2.
- isotope correction as described for Phosphatidylcholine / Sphingomyelin - ESI-MS/MS - Liebisch et al.
Calibration and quantification
- calibration type: matrix calibration - addition of naturally occurring species
- species used for calibration
Species | Cultured cells [pmol] |
---|---|
SPH d18:1 | 30 – 300 |
SPH d18:0 | 15 - 150 |
SPH t18:0 | 10 - 100 |
Dimethyl-SPH d18:1 | 0.3 - 3 |
Trimethyl-SPH d18:1 | 0.3 - 3 |
SPC d18:1 | 10 - 100 |
GluCer d18:1/16:0 | 25 - 250 |
GlaCer d18:1/24:1 | 25 - 250 |
LacCer d18:1/16:0 | 25 - 250 |
LacCer d18:1/24:0 | 25 - 250 |
Cer1P d18:1/16:0 | 15 - 150 |
Cer1P d18:1/24:0 | 15 - 150 |
Method Validation
Accuracy
90 - 110 %
Precision
CV: below 10 %
LOD
below 10 fmol on column, except PhytoSPH 25 fmol and dhCer-1P 50 fmol on column
Recovery
60 - 70%
Sample data
Mass spectra
Chromatogram