Colorectal cancer cell lines show striking isomeric diversity of their O-glycome

Responsible:

Katarina Madunic . LUMC . K.Madunic@lumc.nl

Sample preparation

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1.Sample Origin

General information:
O-linked glycans released from colorectal cancer cell lines

Growth/harvest conditions for recombinantly produced material:
Cells were cultured in Hepes-buffered RPMI 1640 culture medium containing L-glutamine and supplemented with penicillin (5000 IU/ml), streptomycin (5 mg/ml), and 10% (v/v) fetal calf serum (FCS). Cells were incubated at 37 °C with 5% CO2 in humidified air and cell culturing was performed up to a confluence of 80% under sterile conditions. For harvesting of the cells, medium was removed and adherent cells were washed twice with 1x PBS and trypsinized using 1x trypsin/EDTA solution in 1x PBS. To stop trypsin activity, medium in a ratio of 2:5 (trypsin/EDTA/medium; v/v) was added and cells were pelleted at 300 g for 5 min. Cells were then resuspended in 3 ml 1x PBS and counted using the CountessTM Automated Cell Counter (Invitrogen, Paisley, UK) based on tryptan blue staining. Cells were aliquoted to 2.0 106 cells per ml of 1x PBS, and washed twice with 1 ml 1x PBS for 3 min at 1000 g. The supernatant was removed and pellets stored at ?20 °C until further use.

Treatments and/or storage conditions:
Cell pellets were stored at -20C after harvesting before performing analysis.

For commercial material, vendor and applicable item information:
NA

Generation of chemically derived material:
NA

2. Sample Processing

Purification steps:
Cation exchange desalting; PGC SPE

2. Defined Sample

Sample name:
O-linked oligosaccharides

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: Thermo Fisher

Model: Hypercarb

Type of Chromatography: Reverse phase

Type of Material: Carbon

Column diameter: 0,75

Column length: 100

Particle size: 3

Manufacturer url: NA

3. Method run

Temperature: 45 C

Solvent a: 10mM ammonium bicarbonate

Solvent b: 60% ACN, 10mM ammonium bicarbonate

Other solvent: NA

Flow rate: 0,6ul/min

Flow gradient: 2%-50% buffer B

Run time: 73 min

Phase: Reverse

MS

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1. General features

Date stamp: Fri May 11 UTC 2018

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 : Fed

Interface name: NA

Other data for the Interface: NA

Sprayer name: Captive spray

Other data for the sprayer: NA

Relevant voltages: 1000 V

Degree of prompt fragmentation evaluated: No

In-source dissociation performed: Yes

Other parameters if discriminant for the experiment: ACN enriched nebulizing gas (N2)- nanobooster

(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: NA

3. Post-source componentry

Collision-Induced Dissociation - CID

CID. Gas composition: Helium

CID. Gas pressure: NA

CID. Collision energy: 0.27

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