General information:
Novel data acquisition method involving glycan release, optional permethylation, clean-up, and acquisition by MS.
Biologically derived material - Recombinantly produced material
Cell type:
Expi-CHO
Growth/harvest conditions for recombinantly produced material:
Cell lines (Pro5, Lec2, and Lec8) were grown according to the supplier's instructions, in media supplemented with 10% fetal bovine serum. Six cell culture replicates were grown to approximately a 50% density in 6-well plate and allowed to grow for 24–48 h at 37°C until confluent (∼105 cells per well). The medium was aspirated and each well was washed four to five times with PBS. The cell pellets were collected and frozen for downstream preparation. Frozen cells were lysed in 10% SDS via probe sonication (3x 10 seconds on, 10 seconds off)
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:
Human immunoglobulins (IgG, IgM, IgA) were purchased from Athens Research & Technology (https://www.athensresearch.com). SARS-CoV2 spike proteins were produced and sourced from BEI. Purified glycoproteins for initial profiling were purchased from Sigma Aldrich.
Synthesis steps or specify where the equivalent reaction protocol is available:
N-glycan standards were sourced from five vendors (Dextra Laboratories Ltd, Omicron Biochemicals Inc., Glycobia Inc., Chemily LLC, and Cassia LLC). Fresh standards were made up to approximately 50 pmol/µL with ultrapure water with 0.1% piperidine.
Description of starting material:
Free-Oligosaccharides
Enzymatic treatments
Enzymes used for oligosaccharide removal or modification of starting material:
Release method-PNGaseF TREATMENT
Describe vendor or expression and purification procedure:
NEB
Sample material treated in-solution or immobilized? State also temperature, duration, volume, enzyme concentration:
60 µL of PNGase-F digestion solution was added (New England Biolabs, 1 unit, H2O) and incubated at 37 °C for 18 hours.
Chemical treatments
Define the technique for oligosaccharide release or other chemical modifications:
N/A
Reaction conditions (temperature, duration, volume and chemical concentrations):
N/A
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:
N/A
Explain the type of modification employed:
N/A
Source of materials, description of kits used, reaction conditions and detailed workflow:
N/A
Purification steps:
Carbon clean was performed as per manufacturer’s protocol (Hypercarb 25 mg x 96 well plate, Thermo Fisher), which includes loading glycan (0.1% TFA), washing out salts (0.1% TFA), eluting the glycans (50% acetonitrile, 0.1% TFA), and drying down in a 96 well plate.
Sample name:
N-linked oligosaccharides
Manufacturer:
Thermo Fisher
Model:
Vanquish
Instrument details:
Thermofisher Scientific Vanquish Horizon HPLC with MP35N fittings and Vipers.
Manufacturer:
Thermo Fisher
Model:
Hypercarb
Separation Mode:
Reversed phase
Column length:
100 mm
Inner diameter:
1 mm
Total volume (if relevant):
N/A
Stationary Phase:
PGC 3 micron pore size
Column heater:
Vanquish
Additional accessories:
N/A
Description of solvent:
Both mobile phases contained 5 mM HFIP and 5 mM butylamine, with mobile phase A composed of water and mobile phase B composed of 40% water and 60% acetone. LC separation was performed at 155 µL/min.
Description of constituents:
N/A
Time:
20 - 80 min
Gradient:
The final gradient used for glycans released from protein was 0-37% mobile phase B over 70 min, held at 99% B for 5 min, then held at 0% B for 5 min.
Flow rate:
155 ul/min
Temperature:
70 C
Sample injection procedure
Volume:
3-10
Buffer:
H2O
Inline/offline:
Inline
Direct/loop (full/partial loop):
Partial loop
Flush conditions:
Loop kept inline for gradient
Sample storage temperature:
4 oC
Sample loop material:
Stainless steel
Type of sample vials used:
96 well plate
(a) Pre-run process
Type:
Dextran ladder
Substance, standards:
Dextran ladder
Time:
3 injections
Flow rate:
155 ul/min
Type of gradient mixer:
Binary
Post separation events:
Directly into an MS
Column regeneration:
N/A
Equipment used for detection:
MS
Detector type:
MS
Detection system:
MS
Equipment settings
Wavelength(s):
N/A
Gain:
N/A
Frequency that is being detected:
N/A
Sampling frequency (e.g. 10 Hz):
N/A
Timescale over which data was collected:
20-80 min.
(b) Post-run process
Type:
Dextran ladder
Substance, standards:
Dextran ladder
Time:
3 injections
Flow rate:
155 ul/min
Type of gradient mixer:
Binary
Post separation events:
Directly into an MS
Column regeneration:
N/A
Equipment used for detection:
MS
Detector type:
MS
Detection system:
MS
Equipment settings
Wavelength(s):
N/A
Gain:
N/A
Frequency that is being detected:
N/A
Sampling frequency (e.g. 10 Hz):
N/A
Timescale over which data was collected:
N/A
Fraction name:
N/A
(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:
N/A
LC-MALDI spotting:
N/A
Database name:
Glycostore
Database version:
July 24, 2024
Software name:
Skyline
Software version:
25.0
Software URL:
skyline.ms
Peak selection:
Informed based on isotopes and manual integration
Peak quantitation:
Subtraction from noise, trapezoidal intergration
Trace output:
Skyline Assays
Supplier name:
N/A
Exoglycosidase preparation (ref to sample prep):
N/A
Reaction time (ref to sample prep):
N/A
Control:
N/A
Protocol:
N/A
Instrument manufacturer:
Thermo Fisher
Instrument model:
TSQ Altis Plus
Customizations:
N/A
Ion mode:
Negative
Software name:
Xcalibur
Version:
4.6
Upgrades not reflected in version number:
N/A
Switching criteria (tandem only):
N/A
Isolation width (global, or by MS level):
2
Location of ‘parameters’ file:
Raw files, freely viewed by Freestyle
Supply type (static, or fed):
LC
Interface name:
N/A
Catalog number, vendor, and any modifications made to the standard specification:
N/A
Sprayer name:
N/A
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):
Sheath gas at 20 arbitrary units
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:
Argon
Gas pressure:
N/A
Collision energy CID/function:
Varying CE, but beam-type
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):
None
Final MS stage achieved:
2
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:
Quadrupole with Electron Multiplier
UniCarb-DR 
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