General information:
Whole blood from donors with plasma and hemoglobin removed, leaving erythrocytes.
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):
biological fluids
Species:
Homo sapiens (Human)
Treatments and/or storage conditions:
Whole blood was collected from healthy donors, patients with sickle cell disease (SCD), and donors determined to have sickle cell trait (SCT), in EDTA and separated by centrifugation (3500 x g, 10 minutes) to remove plasma. To control for sample age, whole blood was processed seven days post-collection. Plasma was further centrifuged to remove residual red blood cells and fibrinogen (10,000 rpm, 20 minutes) and stored at -20 ºC.
The remaining RBC fraction was further processed to purify erythrocytes and remove hemoglobin. A preliminary separation was performed by diluting the blood equally with DPBS (Dulbecco’s Phosphate Buffered Saline, Gibco/Thermo Fisher Scientific, Waltham, MA) and layering on top of LS Ficoll-Paque Plus (3:4 ratio, GE Healthcare, Chicago, IL). This was centrifuged (400 x g, 30-40 minutes at room temperature (RT), with a low deceleration) and the upper layers of plasma and lymphocytes were removed. Further separation was achieved using 6% dextran, with 1 part dextran, 1 part RBCs, and 2 parts DPBS. The erythrocytes were allowed to rouleaux at RT for 15 minutes. The supernatant was removed and RBCs were washed with DPBS before being lysed with repeated additions of cold MiliQ water and DPBS (pH 8.0) containing protease inhibitor and centrifugation at 4800 x g at 4 ºC until all hemoglobin was removed and a white ghost pellet remained.
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:
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:
Promega
Sample material treated in-solution or immobilized? State also temperature, duration, volume, enzyme concentration:
Overnight incubation (37 °C)
Chemical treatments
Define the technique for oligosaccharide release or other chemical modifications:
PNGase F
Reaction conditions (temperature, duration, volume and chemical concentrations):
N/A
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):
500 mM NaBH4 in a 50 mM KOH solution was added to the membrane spots for 16 h to release reduced O-linked glycans by reductive β-elimination.
Enzymatic modifications
Describe any treatments made to the isolated material:
Release method-PNGaseF TREATMENT
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:
Release method-REDUCTIVE BETA ELIMINATION
Explain the type of modification employed:
Reduction
Source of materials, description of kits used, reaction conditions and detailed workflow:
Released N-glycans were reduced (1 M NaBH4 in 50 mM KOH solution) for 3 hours (50 °C)
Purification steps:
N-glycans were desalted and enriched offline using Dowex 50WX8 (200-400 mesh) strong cation exchange resin (Millipore Sigma, St. Louis, MO) followed by PGC (porous graphitic carbon) solid phase extraction micro-columns (Thermo Fisher Scientific) prior to analysis.
Sample name:
N-linked oligosaccharides
N/A
Instrument manufacturer:
Thermo Fisher
Instrument model:
Scientific LTQ Orbitrap Velos
Customizations:
N/A
Ion mode:
Negative
Software name:
Xcalibur
Version:
2.1
Upgrades not reflected in version number:
N/A
Switching criteria (tandem only):
Top 9 DDA
Isolation width (global, or by MS level):
2 m/z
Location of ‘parameters’ file:
https://panoramaweb.org/GlycoCM.url
Supply type (static, or fed):
LC fed
Interface name:
HESI-II source
Catalog number, vendor, and any modifications made to the standard specification:
IQLAAEGABBFACNMAGY, Thermo Fisher Scientific.
Sprayer name:
Low flow metal insert
Sprayer type, coating, manufacturer, model and catalog number (where available):
Using OPTON-53011 low flow metal needle insert, Thermo Fisher Scientific.
Relevant voltages where appropriate (tip, cone, acceleration):
2700V on tip
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:
Octopoles
Collision-Induced Dissociation (CID)
Gas composition:
Helium (99.999%)
Gas pressure:
N/A
Collision energy CID/function:
33% NCE for resonant CID (RE-CID).
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:
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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