Nanospray ionization mass spectrometry
Mass analysis by NSI-MS are performed by direct infusion of permethylated glycosphingolipids dissolved in infusion buffer the instrument at a constant flow rate of 0.4~1 μL/min via a syringe pump. More detailed procedures are described in Protocol_6 of the sample preparation steps.
General settings
Polarity:
- Positive (for all permethylated GSLs)
- Negative (for charged GSLs those carry strong charge and retains the charge after permethylation, e.g. Sulfatides)
Number of microscans: 3~ 20
Maximum Injection time [ms]: 125~1000
(For MSn acquisition)
Isolation Width [m/z]: 2.2
Normalized Collision Energy: 25~ 45 %*
*Optimum collision energy should be determined experimentally by fragmenting GSL ions.
Thermo Orbitrap-Fusion/Orbitrap-Fusion Lumos
Analyzer: FT, Ion trap
Isolation: Ion trap, Quadrupole*
*Use quadrupole for faster acquisition
Resolution: ~450,000**
**Lower the resolution for faster acquisition
MS/MS activation: CID or HCD for permethylated GSLs
Ion source: EASY-nano spray ion source
Ion Spray Voltage: 1600~2200 V***
***Optimum voltage Should be determined experimentally by looking at the spray stability.
Capillary temperature: 275 oC
Capillary Voltage [V]: Tunes using a permethylated GSL standard
S-lens RF level: 70~120%
Thermo LTQ-Orbitrap-Elite
Analyzer: FT, Ion trap
Isolation: Ion trap
Resolution: ~240,000*
*Lower the resolution for faster acquisition and stronger peak intensity.
MS/MS activation: CID or HCD for permethylated GSLs
Ion source: EASY-nano spray ion source
Ion Spray Voltage: 1.6~2.2 kV*
*Optimum voltage Should be determined experimentally by looking at the spray stability.
Capillary temperature: 275 oC
Capillary Voltage [V]: Tunes using a permethylated GSL standard
S-lens RF level: 70%
Thermo LTQ-Orbitrap XL / LTQ-Orbitrap Discovery
Analyzer: FT, Ion trap
Isolation: Ion trap
Resolution*: ~60,000 (Orbitrap XL) / ~30,000 (Orbitrap Discovery)
*Lower the resolution for faster acquisition
MS/MS activation: CID or HCD (If available) for permethylated GSLs
Ion source: Nano spray ion source
Ion Spray Voltage: 1.6~2.2 kV**
**Optimum voltage Should be determined experimentally by looking at the spray stability.
Capillary temperature: 210 oC
Capillary Voltage [V]: Tunes using a permethylated GSL standard
Tube-lens Voltage: 200-225 V