Binding of Helicobacter pylori to mucin-type fusion proteins carrying Leb, sLex, LDN and GlcNAcα4Gal determinants on different O-glycan core chains

Responsible:

Yolanda H Mthembu . Dept. of Laboratory Medicine, Gothenburg University . yolandamthembu@gmail.com

Sample preparation

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1.Sample Origin

General information:
The CHO-K1 cells were genetically engineered to express a mucin-type fusion protein (PSGL-1/mIgG2b) that carry Leb and SLex determinants on core 2, core 3 and extended core 1 O-glycans. PSGL-1/mIgG2b that carry LacdiNAc on core 2 and the GlcNAcα1, 4Gal1 determinant on core 1 were also expressed by CHO-K1

Growth/harvest conditions for recombinantly produced material:
CHO-K1 cells were seeded in 75-cm2 T-flasks (Nunc, Roskilde, Denmark) in Dulbecco's Modified Eagle's medium (DMEM, Lonza Group Ltd., Basel, Switzerland) supplemented with 10% fetal bovine serum (FBS; Invitrogen AB, Stockholm, Sweden), 2 mM L-glutamine (Invitrogen) and 100 units/mL penicillin, and 100 µg/mL streptomycin (Invitrogen). The cells were maintained at 37oC and 5.0% CO2 in a humidified incubator.

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:
NA

2. Defined Sample

Sample name:
Oligosaccharides

HPLC

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1. General settings

Instrument: Agilent 1100 Series

Manufacturer url: https://www.agilent.com/home

Injector: NA

Injector settings: NA

2. Column

Manufacturer: In-house

Model: Hypercarb

Type of Chromatography: Reversed phase

Type of Material: Porous graphitized carbon

Column diameter: 0.25

Column length: 100

Particle size: 5

Manufacturer url: NA

3. Method run

Temperature: RT

Solvent a: 10 mM NH4HCO3

Solvent b: 0-45% acetonitrile in 10 mM NH4HCO3

Other solvent: NA

Flow rate: 0.010 mL/min

Flow gradient: NA

Run time: 46 min

Phase: Reversed Phase

MS

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1. General features

Date stamp: Mon Jun 17 UTC 2019

Instrument: Thermo Fisher LTQ Linear Ion Trap ESI

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: ESI

Other data for the sprayer: NA

Relevant voltages: NA

Degree of prompt fragmentation evaluated: Yes

In-source dissociation performed: Yes

Other parameters if discriminant for the experiment: NA

(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: NA

CID. Gas pressure: NA

CID. Collision energy: NA

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