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

Announcement date

2021/06/08

Responsible:

Niclas Karlsson . Gothenburg University

Description

Sulfotransferases Gal3ST2, Gal3ST4 and CHST1 add sulfate to core2 glycans on recombinant PSGL-1. O-glycans were analyzed as reduced alditols with porous graphitized carbon chromatography connected to CID/MS/MS in the negative ion mode

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) + sulfotransferases Gal3ST2, Gal3T4 or CHST1

1.1 Biologically derived material

Biologically derived material - Recombinantly produced material

Cell type:
CHO-K1 cells

Growth/harvest conditions for recombinantly produced material:
Transiently expressed core2 transferase (β1,6-N-acetylglucosaminyltransferase 1, GCNT1) and one sulfotransferase (Gal3ST2, Gal3T4 or CHST1)

Biologically derived material - Biological origin of Material

Origin (biological fluid, tissue, etc):
N/A

Species:
N/A

Treatments and/or storage conditions:
N/A

Glycoprotein:
N/A

Biologically derived material - Purchased from commercial manufacturer

Vendor and applicable item information:
N/A

1.2 Chemically derived material

Synthesis steps or specify where the equivalent reaction protocol is available:
N/A

Description of starting material:
N/A

2. Sample Processing

2.1 Sample Processing - Isolation

Chemical treatments

Define the technique for oligosaccharide release or other chemical modifications:
REDUCTIVE BETA ELIMINATION

Reaction conditions (temperature, duration, volume and chemical concentrations):
50 mM NaOH, 0.5M NaBH4, 50 degrees 16 hours

2.2 Sample Processing - Modification

2.3 Sample Processing - Purification

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

3. Defined Sample

Sample name:
O-linked oligosaccharides

Liquid chromatography

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N/A

MS

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1. General features

(a) Global descriptors

Instrument manufacturer:
Thermo Fisher

Instrument model:
LTQ Linear Ion Trap

Customizations:
N/A

Ion mode:
Negative

(b) Control and analysis software

2. Ion sources

(a) Electrospray Ionisation (ESI)

Supply type (static, or fed):
Liquid Chromatograhpy

Interface name:
N/A

Catalog number, vendor, and any modifications made to the standard specification:
Thermo Fisher Electrospray

Sprayer name:
Electrospray

Sprayer type, coating, manufacturer, model and catalog number (where available):
Thermo Fisher metal needle

Relevant voltages where appropriate (tip, cone, acceleration):
Source Voltage 2.5 kV, Capillary Voltage -15 V

Degree of prompt fragmentation evaluated:
Yes

Whether in-source dissociation performed:
No

Other parameters if discriminant for the experiment (such as nebulizing gas and pressure):
N/A

(b) MALDI

Plate composition (or type):
N/A

Matrix composition (if applicable):
N/A

Deposition technique:
N/A

Relevant voltages where appropriate:
N/A

Degree of prompt fragmentation evaluated:
N/A

PSD (or LID/ISD) summary, if performed:
N/A

Operation with or without delayed extraction:
N/A

Laser type (e.g., nitrogen) and wavelength (nm):
N/A

Other laser related parameters, if discriminating for the experiment:
N/A

3. Ion transfer optics

Hardware options:
N/A

(a) Post-source componentry - Collision cell

Collision-Induced Dissociation (CID)

Gas composition:
He

Gas pressure:
N/A

Collision energy CID/function:
Normalised energy 35

Electron Transfer Dissociation (ETD)

Reagent gas:
N/A

Pressure:
N/A

Reaction time:
N/A

Number of reagent atoms:
N/A

Electron Capture Dissociation (ECD)

Emitter type:
N/A

Voltage:
N/A

Current:
N/A

(b) Post-source componentry - TOF drift tube

Reflectron status (on, off, none):
N/A

(c) Post-source componentry - Ion trap

Final MS stage achieved:
MS2

(d) Post-source componentry - Ion mobility

Gas:
N/A

Pressure:
N/A

Instrument-specific parameters:
N/A

(e) Post-source componentry - FT-ICR

Peak selection:
N/A

Pulse:
N/A

Width:
N/A

Voltage:
N/A

Decay time:
N/A

IR:
N/A

Other parameters:
N/A

(f) Post-source componentry - Detectors

Detector type:
Electron multiplier