General information:
N-glycans were released from 10 µL of naked mole-rat plasma by PNGase F after protein denaturation with SDS and Igepal CA-630.
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):
Plasma
Species:
Heterocephalus Glaber
Treatments and/or storage conditions:
Blood was collected on EDTA as anticoagulant and plasma was immediately separated by centrifugation. Samples were stored at -80 °C until analysis.
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:
Glycosidase-PNGASE-F
Describe vendor or expression and purification procedure:
Promega Corp.
Sample material treated in-solution or immobilized? State also temperature, duration, volume, enzyme concentration:
5x PBS, pH 7.4, 18h at 37 °C
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:
Labelling-Reductive Amination
Explain the type of modification employed:
Procainamide (ProA)
Source of materials, description of kits used, reaction conditions and detailed workflow:
2h at 65 °C
Purification steps:
After the reductive amination reaction : HILIC-SPE on GHP membrane (hydrophilic polypropylene) was used to purify released and ProA-labeled N-glycans. Afterwards, labeled N-glycans were fractionated on weak anion exchange column (WAX) into five fractions. Each fraction was dried down and desalted on porous graphitic carbon (PGC) StageTips.
Sample name:
N-linked oligosaccharides
Manufacturer:
Waters
Model:
H-class
Instrument details:
Waters Acquity H-class
Manufacturer:
Waters
Model:
Waters XBridge BEH amide column
Separation Mode:
HILIC
Column length:
150 mm
Inner diameter:
0.2 mm
Total volume (if relevant):
N/A
Stationary Phase:
Amide HILIC
Column heater:
Yes. Heated to 50 °C.
Additional accessories:
N/A
Description of solvent:
A: 100 mM ammonium formate, pH 4.4, in ultrapure water
B: acetonitrile, LC-MS grade
Description of constituents:
N/A
Time:
90 min
Gradient:
25-40 % Solvent A in 90 min
Flow rate:
0.5 mL/min
Temperature:
50 °C
Sample injection procedure
Volume:
40 µL
Buffer:
75 % ACN in 100 mM ammonium formate, pH 4.4
Inline/offline:
Inline
Direct/loop (full/partial loop):
Partial loop
Flush conditions:
N/A
Sample storage temperature:
10 °C
Sample loop material:
Stainless steel
Type of sample vials used:
PP vials
(a) Pre-run process
Type:
N/A
Substance, standards:
N/A
Time:
N/A
Flow rate:
N/A
Type of gradient mixer:
N/A
Post separation events:
N/A
Column regeneration:
N/A
Equipment used for detection:
FLR
Detector type:
FLR
Detection system:
Waters FLR detector
Equipment settings
Wavelength(s):
Ex/Em 310/370 nm
Gain:
1.0
Frequency that is being detected:
2 pts/sec
Sampling frequency (e.g. 10 Hz):
2 pts/sec
Timescale over which data was collected:
90 min
(b) Post-run process
Type:
N/A
Substance, standards:
N/A
Time:
N/A
Flow rate:
N/A
Type of gradient mixer:
N/A
Post separation events:
N/A
Column regeneration:
N/A
Equipment used for detection:
N/A
Detector type:
N/A
Detection system:
N/A
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:
N/A
Database version:
N/A
Software name:
GlycoWorkbench
Software version:
2.1
Software URL:
https://code.google.com/archive/p/glycoworkbench/
Peak selection:
Manual
Peak quantitation:
Manual integration
Trace output:
N/A
Supplier name:
Promega
Exoglycosidase preparation (ref to sample prep):
in 5x PBS, pH 7.4
Reaction time (ref to sample prep):
18h
Control:
N/A
Protocol:
N/A
Instrument manufacturer:
Bruker Daltonik
Instrument model:
compact
Customizations:
N/A
Ion mode:
Positive
Software name:
Compass
Version:
3.2
Upgrades not reflected in version number:
SR 2, Build 44
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):
Fed
Interface name:
IonBooster
Catalog number, vendor, and any modifications made to the standard specification:
Bruker
Sprayer name:
IonBooster
Sprayer type, coating, manufacturer, model and catalog number (where available):
N/A
Relevant voltages where appropriate (tip, cone, acceleration):
Capillary: 2250 V; Endplate offset: -750 V; ionBooster charging voltage: 300 V
Degree of prompt fragmentation evaluated:
No
Whether in-source dissociation performed:
No
Other parameters if discriminant for the experiment (such as nebulizing gas and pressure):
Nebulizer: 5.5 bar; Drying gas: 4L/min; Temperature: 200 °C. Nitrogen.
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:
Ion funnels, hexapole, quadrupole, CC, TOF
Collision-Induced Dissociation (CID)
Gas composition:
Argon, purity 9.0
Gas pressure:
25 %
Collision energy CID/function:
Acquisition Parameter Value _x000D_
Collision Energy 5.0 eV _x000D_
Collision Gas Supply Flowrate 25.0 % _x000D_
Set QuenchTime 5 ms _x000D_
Set Quench Enable On _x000D_
Set Quench Always Enable On _x000D_
Set Collision Gas Supply Switch On _x000D_
Set Collision Stepping Active On _x000D_
Set Collision Stepping Mode Basic _x000D_
Set Collision Cell Entrance Fix On _x000D_
Set Focus 2 - Lens 1 27.8 V _x000D_
Set Focus 2 - Lens 2 -85.0 V _x000D_
Set Focus 2 - Lens 3 29.8 V _x000D_
Set Collision Cell Bias 25.0 V _x000D_
Set Collision Cell RF 2100.0 Vpp _x000D_
Set Collision Storage 50.0 V _x000D_
Set Collision Extraction 20.0 V _x000D_
Set Transfer Time 150.0 µs _x000D_
Set PrePulseStorage Time 10.0 µs _x000D_
Set Focus 3 - Lens 2 0.0 V _x000D_
Set Focus 3 - Lens 3 -100.0 V _x000D_
Set Focus 3 - Lens 4 0.0 V _x000D_
Set Focus 3 - Lens 5 -36.5 V _x000D_
Set Collision Energy Offset 5.0 eV _x000D_
Set Collision Energy ( MS only ) 5.0 eV _x000D_
Set Collision Gas Supply Flowrate 25.0 % _x000D_
Set Collision Stepping Switch Time(s) 0; 25; 50; 75 % _x000D_
Set Collision Stepping RF Factor(s) 1.0000; 1.0000; 1.0000; 1.0000 _x000D_
Set Collision Stepping Energy Factor(s) 1.000; 1.000; 1.000; 1.000 _x000D_
Set Collision Stepping Transfer Time Factor(s) 0.471; 0.471; 1.000; 1.000 _x000D_
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):
On
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 
Supported by SIB ExPASy | JST / NBDCDICP
