O-glycans released from platelet releasate samples activated with thrombin (0.2 U/mL)

Announcement date

2022/09/25

Responsible:

Dr Morten Thaysen-Andersen . Macquarie University

Description

O-glycans released from platelet releasate samples activated with thrombin (0.2 U/mL)

Sample preparation

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1. Sample Origin

General information:
O-glycans released from platelet releasate samples activated with thrombin (0.2 U/mL)

1.1 Biologically derived material

Biologically derived material - Recombinantly produced material

Cell type:
N/A

Growth/harvest conditions for recombinantly produced material:
N/A

Biologically derived material - Biological origin of Material

Origin (biological fluid, tissue, etc):
biological fluids

Species:
Homo sapiens (Human)

Treatments and/or storage conditions:
-80°C

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:
Glycoproteins

2. Sample Processing

2.1 Sample Processing - Isolation

Enzymatic treatments

Enzymes used for oligosaccharide removal or modification of starting material:
Release method-PNGaseF TREATMENT

Describe vendor or expression and purification procedure:
Promega

Sample material treated in-solution or immobilized? State also temperature, duration, volume, enzyme concentration:
2 U per 20 ug of sample, incubation overnight at 37°C

Chemical treatments

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

Reaction conditions (temperature, duration, volume and chemical concentrations):
500 mM NaBH4 in 50 mM KOH solution was added to immobilised protein spots for 16 h at 50˚C

2.2 Sample Processing - Modification

Enzymatic modifications

Describe any treatments made to the isolated material:
PNGase F

Enzyme concentration, supplier, biological source, incubation time and temperature:
2 U per 20 ug of sample, incubation overnight at 37°C

If novel glycosidase was used, provide information indicating the origin (i.e. species) of the enzyme:
Promega

Chemical modifications

Describe any treatments made to the isolated material:
Labelling-REDUCTION

Explain the type of modification employed:
Reduction of reducing end

Source of materials, description of kits used, reaction conditions and detailed workflow:
1 M NaBH4 in 50 mM KOH solution, 3 h at 50 °C, followed by neutralisation using equimolar of glacial acetic acid

2.3 Sample Processing - Purification

Purification steps:
Dual desalting of the reduced O-glycans was performed using firstly strong cation exchange resin (AG 50W-X8 Resin, Bio-Rad) followed by porous graphitised carbon (PGC) resin custom packed as micro-columns on top of C18 discs (Merck-Millipore) in P10 solid-phase extraction (SPE) formats.

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

Customizations:
N/A

Ion mode:
Negative

(b) Control and analysis software

Software name:
Xcalibur

Version:
2.2

Upgrades not reflected in version number:
N/A

Switching criteria (tandem only):
N/A

Isolation width (global, or by MS level):
0.5 m/z

Location of ‘parameters’ file:
N/A

2. Ion sources

(a) Electrospray Ionisation (ESI)

Supply type (static, or fed):
PGC-LC-MS

Interface name:
N/A

Catalog number, vendor, and any modifications made to the standard specification:
N/A

Sprayer name:
Low flow metal insert

Sprayer type, coating, manufacturer, model and catalog number (where available):
N/A

Relevant voltages where appropriate (tip, cone, acceleration):
N/A

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):
A full scan acquisition range of m/z 300-2,000, a resolution of m/z 0.25 full width half maximum and a source voltage of +3.2 kV. The automatic gain control for the MS1 scans was set to 5 x 104 with a maximum accumulation time of 50 ms. For the MS/MS events, the resolution was set to m/z 0.35 full width half maximum, the automatic gain control was 2 x 104 and the maximum accumulation time was 300 ms.

(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:
Helium

Gas pressure:
N/A

Collision energy CID/function:
Collision was performed at 35 % normalised collision energy and an isolation window of 4 m/z

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:
N/A

(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:
N/A