General information:
Investigation of the Gilthead seabreams intestines mucus glycosylation in response to parasites.
Biologically derived material - Recombinantly produced material
Cell type:
N/A
Growth/harvest conditions for recombinantly produced material:
-
Biologically derived material - Biological origin of Material
Origin (biological fluid, tissue, etc):
biological fluids
Species:
Sparus aurata
Treatments and/or storage conditions:
-80 C.
Glycoprotein:
-
Biologically derived material - Purchased from commercial manufacturer
Vendor and applicable item information:
-
Synthesis steps or specify where the equivalent reaction protocol is available:
-
Description of starting material:
Glycoproteins
Enzymatic treatments
Enzymes used for oligosaccharide removal or modification of starting material:
Release method-REDUCTIVE BETA ELIMINATION
Describe vendor or expression and purification procedure:
-
Sample material treated in-solution or immobilized? State also temperature, duration, volume, enzyme concentration:
-
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):
50 C
Enzymatic modifications
Describe any treatments made to the isolated material:
Release method-REDUCTIVE BETA ELIMINATION
Enzyme concentration, supplier, biological source, incubation time and temperature:
-
If novel glycosidase was used, provide information indicating the origin (i.e. species) of the enzyme:
-
Chemical modifications
Describe any treatments made to the isolated material:
Release method-REDUCTIVE BETA ELIMINATION
Explain the type of modification employed:
-
Source of materials, description of kits used, reaction conditions and detailed workflow:
-
Purification steps:
-
Sample name:
Oligosaccharides
Manufacturer:
Thermo Fisher
Model:
Vanquish_Neo
Instrument details:
-
Manufacturer:
In-house
Model:
Custom made Hypercarb
Separation Mode:
-
Column length:
~10 cm
Inner diameter:
250 um
Total volume (if relevant):
N/A
Stationary Phase:
PGC
Column heater:
-
Additional accessories:
-
Description of solvent:
Water-Acetonitrile
Description of constituents:
N/A
Time:
68 min
Gradient:
0-40 Acetonitrile
Flow rate:
10 ul/min
Temperature:
20
Sample injection procedure
Volume:
2 ul
Buffer:
10 mM ammonium bicarbonate
Inline/offline:
-
Direct/loop (full/partial loop):
-
Flush conditions:
-
Sample storage temperature:
-
Sample loop material:
-
Type of sample vials used:
-
(a) Pre-run process
Type:
-
Substance, standards:
-
Time:
-
Flow rate:
-
Type of gradient mixer:
-
Post separation events:
-
Column regeneration:
-
Equipment used for detection:
-
Detector type:
-
Detection system:
-
Equipment settings
Wavelength(s):
-
Gain:
-
Frequency that is being detected:
--
Sampling frequency (e.g. 10 Hz):
-
Timescale over which data was collected:
-
(b) Post-run process
Type:
-
Substance, standards:
-
Time:
-
Flow rate:
-
Type of gradient mixer:
-
Post separation events:
-
Column regeneration:
-
Equipment used for detection:
-
Detector type:
-
Detection system:
-
Equipment settings
Wavelength(s):
-
Gain:
-
Frequency that is being detected:
-
Sampling frequency (e.g. 10 Hz):
-
Timescale over which data was collected:
-
Fraction name:
-
(a) Description of the procedure by which the fractions were collected
Start time:
N/A
End time:
N/A
Mode (fixed or peak directed):
N/A
(b) Description of the individual fractions
Time of collection:
N/A
Volume:
N/A
Charge:
N/A
Flow split:
-
LC-MALDI spotting:
-
Database name:
-
Database version:
-
Software name:
Xcalibur
Software version:
2.2
Software URL:
-
Peak selection:
-
Peak quantitation:
-
Trace output:
-
Supplier name:
-
Exoglycosidase preparation (ref to sample prep):
-
Reaction time (ref to sample prep):
-
Control:
-
Protocol:
-
Instrument manufacturer:
Thermo Fisher
Instrument model:
LTQ Linear Ion Trap
Customizations:
N/A
Ion mode:
Negative
Software name:
Xcalibur
Version:
2.2
Upgrades not reflected in version number:
N/A
Switching criteria (tandem only):
MS2 triggered from the survey scan of the 2 most intense peaks > 200 in intensity
Isolation width (global, or by MS level):
m/z 3.0
Location of ‘parameters’ file:
N/A
Supply type (static, or fed):
fed
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
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
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:
Helium
Gas pressure:
N/A
Collision energy CID/function:
0.35
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
Reflectron status (on, off, none):
N/A
Final MS stage achieved:
MS2
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:
Electron multiplier
UniCarb-DR 
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