General information:
Cells
Growth/harvest conditions for recombinantly produced material:
Growth conditions: Briefly, cell lines were cultured in DMEM medium supplemented with 10% FBS and 1% antibiotics (Streptomycin and Penicillin) at 37 °C and 5% CO2. Harvest conditions: The cells were harvested at around 80% confluence and detached with trypsin followed by washed with 5ml of 1 x PBS for 3 times.
Treatments and/or storage conditions:
NA
For commercial material, vendor and applicable item information:
NA
Generation of chemically derived material:
NA
Purification steps:
Purification of GSLs extracted from cells: The tC18 RP-SPE cartridge was preconditioned by the sequential passing of 1 mL of chloroform/MeOH (2:1), 1 mL MeOH and 2 mL MeOH/H2O (1:1) over the cartridge. The extracted GSLs were resuspended in 200 µL using a mixture of MeOH and H2O (1:1) and loaded on the tC18 RP-SPE cartridge by passing the sample three times over the cartridge. The loaded cartridge was washed with 2 mL of MeOH/H2O (1:1), followed by a sequential elution of the GSLs using 2 mL of MeOH and 2 mL of chloroform/MeOH (2:1). The samples were dried under vacuum in an Eppendorf Concentrator at 30 °C. Purification of released GSLs glycans: the GSL glycans were retrieved by using a tC18 RP-SPE cartridge, which was preconditioned with 2 mL of MeOH and 2 mL of H2O prior to applying the sample onto the cartridge. The purified GSL glycans were eluted by using 500 µL of H2O. The mixture, consisting of the elution and flowthrough, was lyophilized under vacuum. Purification of reduced GSL glycans: Lyophilized GSL glycans were reconstituted in 40 µL of H2O with 0.1% TFA (v/v), followed by a shaking step for 30 min. PGC SPE clean-up was performed as described previously with some slight modifications. Briefly, columns were packed with 50 µL of PGC resin slurry (containing approximately 25 µg of PGC material), followed by a conditioning step by loading 3 x 60 µL of 80% MeCN with 0.1% TFA (v/v) followed by 3 x 60 µL of H2O with 0.1% TFA (v/v). After sample loading, the columns were washed with 2 x 60 µL of H2O with 0.1% TFA (v/v). Subsequently, the GSL glycans were eluted by adding 2 x 40 µL of 60% MeCN with 0.1% TFA (v/v). The two elution fractions were combined and dried under vacuum.
Sample name:
Glycolipids
Instrument: Thermo Fisher 1100 Series
Manufacturer url: NA
Injector: DioneX autosampler
Injector settings: NA
Manufacturer: In-house
Model: Hypercarb
Type of Chromatography: Reverse phase
Type of Material: Porous graphitized carbon
Column diameter: 75 um
Column length: 10 cm
Particle size: 3 um
Manufacturer url: NA
Temperature: 35°C
Solvent a: 10mM ammonium bicarbonate
Solvent b: 60% ACN, 10mM ammonium bicarbonate
Other solvent: NA
Flow rate: 0,6ul/min
Flow gradient: 1%-50% buffer B
Run time: 73min
Phase: Reverse
Date stamp: Sun Nov 14 UTC 2021
Instrument: Bruker Daltonik amaZon Speed ETD ETD
Customizations from the manufacturer's specification:
NA
ion_mode: -
Sprayer type : nanoflow LC/MS
Interface name: NA
Other data for the Interface: Bruker Daltonik
Sprayer name: Captive spray
Other data for the sprayer: NA
Relevant voltages: 1000V
Degree of prompt fragmentation evaluated: Yes
In-source dissociation performed: Yes
Other parameters if discriminant for the experiment: 2-propanol enriched nebulizing gas (N2)- nanobooster with dry gas temperature 280°C , 5 L/min and pressure 3 psi
Plate composition (or type): NA
Matrix composition: NA
Deposition technique: NA
Relevant voltages: NA
Degree of prompt fragmentation evaluated: NA
PSD (or LID/ISD) summary: NA
Operation with or without delayed extraction: NA
Laser type: NA
Other laser related parameters: NA
url: NA
Hardware options: Amazon speed CID
CID. Gas composition: Helium
CID. Gas pressure: NA
CID. Collision energy: Smart Frag with amplitude 29-120% within 32 ms with a default cut off
ETD. Reagent gas: NA
ETD. Pressure: NA
ETD. Reaction time: NA
ETD. Reagent atoms: NA
ECD. Emitter type: NA
ECD. Voltage: NA
ECD. Current: NA
TOF. Reflectron status: NA
Ion trap. Final MS stage achieved: NA
Ion mobility. Gas type : NA
Ion mobility. Pressure: NA
Ion mobility. Instrument parameters: NA
Peak selection: NA
FT-ICR. Pulse: NA
FT-ICR. Width: NA
FT-ICR. IR: Decay time: NA
FT-ICR. IR: NA
FT-ICR. Other parameters: NA
Detector type: NA