General information:
Protein glycosylation transformations such as the ones of O-glycans are known to be associated with the pathogenesis of many cancer types. This is largely attributed to differential glycosyltransferase expression or activity. These alterations affect the biological function and protein integrity, which directly affects cancer growth and progression. There is still a lack of knowledge about the head and neck squamous cell carcinoma (HNSCC) O-glycome, hence we attempted to capture the HNSCC tumour tissue O-glycome using the Porous Graphitized Carbon Liquid Chromatography coupled to Electrospray Ionization Tandem Mass Spectrometry glycomics approach. We identified that sialylated O-glycans are highly abundant in HNSCC tissue, with the sialylated and di-sialylated T-antigens being the most abundant O-glycan in both tumour and surrounding tissue. Interruption of sialylation by both, sialyltransferase inhibitors and neuraminidases significantly inhibited migration of HNSCC cells without affecting their proliferation, indicating a vital role of sialylation for HNSCC cell migration.
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):
tissue
Species:
Homo sapiens (Human)
Treatments and/or storage conditions:
Tumour tissue were fixed onto a PEN membrane glass slides
Glycoprotein:
N/A
Biologically derived material - Purchased from commercial manufacturer
Vendor and applicable item information:
RBWH Hospital, Queensland Australia
Synthesis steps or specify where the equivalent reaction protocol is available:
HNC FFPE tumour tissue sections (10 μm thick) were deposited onto a PEN membrane glass slide. A total of 8 patient FFPE tumour and normal tissues were collected between 2018-2020 from Royal Brisbane and Women’s hospital, Queensland, Australia. From these tissue sections it was possible to isolate matching normal tissue for four patients (CTC16, CTC27, CTC59, CTC40). To reduce any possible gender-derived heterogeneity just tissue sections from male patients were obtained. Tumor and normal epithelial tissue regions were annotated by an expert pathologist from H&E-stained slides. Hematoxylin and eosin-stained slides were used for pathologist annotation. Slides used for sample processing were deparaffinised with xylene (washed thrice for 5 mins) followed by two washes with absolute ethanol for 5 mins. Tissues were collected into individual Eppendorf tubes using a scalpel according to the pathologist’s annotation. 50 µl of 0.1 M Tris-HCl (pH 8.0) and 10 µl of 0.1 M DDT were added to each sample before they were sonicated for 30 seconds three times and SDS was added to reach the final concentration of 4 % (w/v). Samples were heated to 99 oC for 1 hour with mild agitation. Samples were then centrifuged for 20 mins at 2000 rcf and the supernatant was collected for protein precipitation, which was performed by adding 4 times methanol, an equal volume of chloroform, three times water before the entire mixture was vigorously vortexed for 30 seconds. The samples were centrifuged at 14,000 rcf for 5 min and the supernatant was carefully discarded (upper aqueous phase). Additional methanol was added, mixed properly by brief vortexing and centrifuged for 10 min at 14,000 rcf. The supernatant was discarded, and the final pellet was dissolved in a solution containing 6 M urea and 2 M thiourea.
Description of starting material:
Free-Oligosaccharides
Enzymatic treatments
Enzymes used for oligosaccharide removal or modification of starting material:
N/A
Describe vendor or expression and purification procedure:
N/A
Sample material treated in-solution or immobilized? State also temperature, duration, volume, enzyme concentration:
N/A
Chemical treatments
Define the technique for oligosaccharide release or other chemical modifications:
Xylene, Tris-HCl (pH 8.0) and 10 µl of 0.1 M DDT
Reaction conditions (temperature, duration, volume and chemical concentrations):
N/A
Enzymatic modifications
Describe any treatments made to the isolated material:
PNGase F
Enzyme concentration, supplier, biological source, incubation time and temperature:
Overnight digestion
If novel glycosidase was used, provide information indicating the origin (i.e. species) of the enzyme:
New England Biolabs (VIC)
Chemical modifications
Describe any treatments made to the isolated material:
Release method-REDUCTIVE BETA ELIMINATION
Explain the type of modification employed:
Alkaline condition
Source of materials, description of kits used, reaction conditions and detailed workflow:
16 hours at 50 degree celcious
Purification steps:
Porous graphitic carbon clean up
Sample name:
O-linked oligosaccharides
N/A
Instrument manufacturer:
Bruker Daltonik
Instrument model:
6340 Ion Trap LC/MS
Customizations:
N/A
Ion mode:
Negative
Software name:
DataAnalysis
Version:
4.2
Upgrades not reflected in version number:
N/A
Switching criteria (tandem only):
N/A
Isolation width (global, or by MS level):
N/A
Location of ‘parameters’ file:
N/A
Supply type (static, or fed):
CAPTIVE SPRAY
Interface name:
N/A
Catalog number, vendor, and any modifications made to the standard specification:
N/A
Sprayer name:
CID, ESI Nano spray
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:
N/A
Other parameters if discriminant for the experiment (such as nebulizing gas and pressure):
N/A
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
Hardware options:
N/A
Collision-Induced Dissociation (CID)
Gas composition:
nitrogen drying gas
Gas pressure:
12 psi
Collision energy CID/function:
N/A
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:
CID
Voltage:
N/A
Current:
N/A
Reflectron status (on, off, none):
N/A
Final MS stage achieved:
N/A
Gas:
N/A
Pressure:
N/A
Instrument-specific parameters:
N/A
Peak selection:
N/A
Pulse:
N/A
Width:
N/A
Voltage:
N/A
Decay time:
N/A
IR:
N/A
Other parameters:
N/A
Detector type:
N/A
UniCarb-DR 
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