Sulfation of O-glycans on mucin-type proteins from serous ovarian epithelial tumors

Responsible:

Kristina Thomsson . Institute of Biomedicine, University of Gothenburg . kristina.thomsson@medkem.gu.se

Sample preparation

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1.Sample Origin

General information:
Recombinant human PSGL-1/mIgG2b purified from CHO cells with transiently expressed core2 transferase (β1,6-N-acetylglucosaminyltransferase 1, GCNT1) + sulfotransferase Gal3ST2

Growth/harvest conditions for recombinantly produced material:
Recombinant human PSGL-1/mIgG2b purified from CHO cells with transiently expressed core2 transferase (β1,6-N-acetylglucosaminyltransferase 1, GCNT1) + sulfotransferase Gal3ST2

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:
After reductive b elimination, salts are removed by cation exchange beads packed in zip tip pipets, eluted with water, samples were lyphilized and borate complexes removed by repeated MeOH addition/evaporation

2. Defined Sample

Sample name:
O-linked oligosaccharides

HPLC

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1. General settings

Instrument: Agilent 1100 Series

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

Injector: Pal systems - HTC Pal

Injector settings: 3 ul drawn injected into a 2 ul loop

2. Column

Manufacturer: In-house

Model: NA

Type of Chromatography: Graphitized carbon

Type of Material: Graphitized carbon

Column diameter: 250 um

Column length: 10 cm

Particle size: 10 u

Manufacturer url: NA

3. Method run

Temperature: RT

Solvent a: 10 mM (NH4)HCO3

Solvent b: 10 mM (NH4)HCO3 in 80% acetonitrile

Other solvent: NA

Flow rate: approx 3-5 ul/min (static split from 250 ul/min)

Flow gradient: 0-50% B in 40 min, 50-100% B from 40-48 min

Run time: 78 min

Phase: Reversed phase

MS

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1. General features

Date stamp: Fri Jun 04 UTC 2021

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 : Liquid Chromatograhpy

Interface name: NA

Other data for the Interface: Thermo Fisher Electrospray

Sprayer name: Electrospray

Other data for the sprayer: Thermo Fisher metal needle

Relevant voltages: Source Voltage 2.5 kV, Capillary Voltage -15 V

Degree of prompt fragmentation evaluated: Yes

In-source dissociation performed: No

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: He

CID. Gas pressure: NA

CID. Collision energy: Normalised energy 35

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: MS2

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: Electron multiplier