A-series gangliosides inhibit TGF-β-induced epithelial-mesenchymal transition via TRI degradation

Responsible:

Tao Zhang . LUMC . t.zhang@lumc.nl

Sample preparation

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1.Sample Origin

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

2. Sample Processing

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.

2. Defined Sample

Sample name:
Glycolipids

HPLC

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1. General settings

Instrument: Thermo Fisher 1100 Series

Manufacturer url: NA

Injector: DioneX autosampler

Injector settings: NA

2. Column

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

3. Method run

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

MS

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1. General features

Date stamp: Fri Jul 10 UTC 2020

Instrument: Bruker Daltonik amaZon Speed ETD ETD

Customizations from the manufacturer's specification:
NA

ion_mode: -

2. Ion sources

(a) Electrospray ionisation - ESI

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

(b) MALDI

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

3. Ion transfer optics

Hardware options: Amazon speed CID

3. Post-source componentry

Collision-Induced Dissociation - 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

Electron Transfer Dissociation - ETD

ETD. Reagent gas: NA

ETD. Pressure: NA

ETD. Reaction time: NA

ETD. Reagent atoms: NA

Electron Capture Dissociation - ECD

ECD. Emitter type: NA

ECD. Voltage: NA

ECD. Current: NA

TOF drift tube

TOF. Reflectron status: NA

Ion Trap

Ion trap. Final MS stage achieved: NA

Ion mobility

Ion mobility. Gas type : NA

Ion mobility. Pressure: NA

Ion mobility. Instrument parameters: NA

FT-ICR

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

Detectors

Detector type: NA