General information:
Glycoproteins from barramundi mucus (Lates Calcarifer)
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):
Mucus(skin, gill, mouth, intestine)
Species:
Lates calcarifer
Treatments and/or storage conditions:
N/A
Glycoprotein:
N/A
Biologically derived material - Purchased from commercial manufacturer
Vendor and applicable item information:
N/A
Synthesis steps or specify where the equivalent reaction protocol is available:
N/A
Description of starting material:
N/A
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:
REDUCTIVE BETA ELIMINATION
Reaction conditions (temperature, duration, volume and chemical concentrations):
50 mM NaOH, 0.5M NaBH4, 50 degrees 16 hours
Enzymatic modifications
Describe any treatments made to the isolated material:
N/A
Enzyme concentration, supplier, biological source, incubation time and temperature:
N/A
If novel glycosidase was used, provide information indicating the origin (i.e. species) of the enzyme:
N/A
Chemical modifications
Describe any treatments made to the isolated material:
REDUCTION
Explain the type of modification employed:
Reduced Reducing end
Source of materials, description of kits used, reaction conditions and detailed workflow:
N/A
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
Sample name:
O-linked oligosaccharides
Manufacturer:
Thermo Fisher
Model:
Vanquish Neo
Instrument details:
Vanquish Neo
Manufacturer:
In-house
Model:
Custom made Hypercarb
Separation Mode:
Reversed phase
Column length:
10 cm
Inner diameter:
250 um
Total volume (if relevant):
N/A
Stationary Phase:
Porous graphitized carbon
Column heater:
No
Additional accessories:
No
Description of solvent:
Buffer A: 10 mM ammonium bicarbonate
Buffer B: 10 mM ammonium bicarbonate 80% acetonitrile
Description of constituents:
N/A
Time:
52 min
Gradient:
1-25% buffer B 5-20min
Flow rate:
6 ul/min
Temperature:
na
Sample injection procedure
Volume:
2 ul
Buffer:
water
Inline/offline:
inline
Direct/loop (full/partial loop):
partial loop
Flush conditions:
na
Sample storage temperature:
-20C
Sample loop material:
na
Type of sample vials used:
na
(a) Pre-run process
Type:
na
Substance, standards:
na
Time:
na
Flow rate:
na
Type of gradient mixer:
na
Post separation events:
na
Column regeneration:
na
Equipment used for detection:
na
Detector type:
na
Detection system:
na
Equipment settings
Wavelength(s):
na
Gain:
na
Frequency that is being detected:
na
Sampling frequency (e.g. 10 Hz):
na
Timescale over which data was collected:
na
(b) Post-run process
Type:
na
Substance, standards:
na
Time:
na
Flow rate:
na
Type of gradient mixer:
na
Post separation events:
na
Column regeneration:
na
Equipment used for detection:
na
Detector type:
na
Detection system:
na
Equipment settings
Wavelength(s):
na
Gain:
na
Frequency that is being detected:
na
Sampling frequency (e.g. 10 Hz):
na
Timescale over which data was collected:
na
Fraction name:
na
(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
Flow split:
na
LC-MALDI spotting:
na
Database name:
na
Database version:
na
Software name:
na
Software version:
na
Software URL:
na
Peak selection:
na
Peak quantitation:
na
Trace output:
na
Supplier name:
na
Exoglycosidase preparation (ref to sample prep):
na
Reaction time (ref to sample prep):
na
Control:
na
Protocol:
na
Instrument manufacturer:
Thermo Fisher
Instrument model:
Fusion orbitrap
Customizations:
N/A
Ion mode:
Negative
Software name:
Xcalibur
Version:
4.6
Upgrades not reflected in version number:
N/A
Switching criteria (tandem only):
MSn of selected precursors
Isolation width (global, or by MS level):
m/z 2.0
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 or MS3
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 
Supported by SIB ExPASy | JST / NBDCDICP
