General information:
Released N- and O-glycans released from glycoprotein standards, cell line lysates, secreted protein from cell lines and mouse tissue mixed with dextran ladder internal standard before PGC-LC-ESI-MS/MS analysis.
Biologically derived material - Recombinantly produced material
Cell type:
macrophage
Growth/harvest conditions for recombinantly produced material:
The U87MG, HEK293 and BV2 cell lines were cultured in T-75 flasks with 10 mL of Dulbecco’s Modified Eagle’s Medium supplemented with 10% (v/v) fetal bovine serum. The PC-12 cell line was cultured in similar conditions with slight modifications, using 10 mL of RPMI-1640 Medium supplemented with 10% (v/v) heat-inactivated horse serum, 5% (v/v) fetal bovine serum and 50ng/mL neural growth factor. After the adherent cells reached 70% confluency, the growth medium was removed and adherent cells scraped and collected. The cells were washed with phosphate buffered saline, collected by centrifugation(500 g for 10 min), and supernatant was removed for a total of three washes. Following cell lysis, proteins were precipitated using chloroform:methanol:water extraction (10:10:3, by volume). The protein pellet was collected re-solubilized in 4 M urea, and quantified by the Bradford protein assay.
For analysis of secreted proteins, EX-CELL 293 (Sigma Aldrich, Sydney, Australia) was reconstituted as specified by the manufacturer. Once cells reached 70% confluency in the standard culture media containing serum, the culture medium was removed, and cells were
View Article Online DOI: 10.1039/C9AN00486F
washed three times with phosphate buffered saline (PBS) before 10mL of the reconstituted EX-CELL 293 medium was added to the cells. Cells were acclimatized for 48 hr before secreted protein collection. Secreted proteins were precipitated using a final concentration of 90% (v/v) acetone, solubilized and quantified as described above.
Biologically derived material - Biological origin of Material
Origin (biological fluid, tissue, etc):
tissue
Species:
Mus musculus (Mouse)
Treatments and/or storage conditions:
Pathogen-free adult male Balb/c mice (300–350 g; University of Adelaide, Australia, approval number: M-2013-227) were deeply anesthetized using an i.p. injection of 60mg/kg Lethabarb and then transcardially perfused using cold 0.9% saline. The lumbar enlargement (L3-L5) of the spinal cord was removed; snap frozen and stored at -80°C until further assessment. A similar method was used for brain sectioning with the brain quickly removed, snap frozen, periaqueductal grey (PAG) and rostral ventromedulla (RVM) containing regions sliced in 50 μm sections in a cryostat and stored at 80 °C until further assessment.
Glycoprotein:
N/A
Biologically derived material - Purchased from commercial manufacturer
Vendor and applicable item information:
Bovine ribonuclease B (product#:R7884), porcine gastric mucin (product#:M1778), human IgA (product#:I1010), bovine lactoferrin (product#:L9507), human lactoferrin (product#:L0520), bovine fetuin (product#:F3385) and human IgG (product#:I4506) were sourced from Sigma Aldrich (Sydney, Australia). Human neutrophil elastase (product#:342-40) was sourced from LeeBio (Maryland Heights, USA).
Synthesis steps or specify where the equivalent reaction protocol is available:
10 mg of dextran T2000 (Amersham Pharmacia Biotech AB, Uppsala, Sweden) was dissolved in 1 mL of 1 M trifluoroacetic acid (TFA) and held at 80 ˚C for 30 min. After this partial acid hydrolysis, the sample was dried by centrifugal evaporation at room temperature.
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 (Sydney, Australia)
Sample material treated in-solution or immobilized? State also temperature, duration, volume, enzyme concentration:
20ug of protein with 1 unit PNGase-F at 37 °C, overnight
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 50 mM KOH solution was added to immobilized protein spots for 16 h at 50 ˚C
Enzymatic modifications
Describe any treatments made to the isolated material:
Release method-PNGaseF TREATMENT
Enzyme concentration, supplier, biological source, incubation time and temperature:
Described above.
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-REDUCTION
Explain the type of modification employed:
Reduction
Source of materials, description of kits used, reaction conditions and detailed workflow:
1 M NaBH4 in 50 mM KOH solution which was held at 50 ˚C for 3 hours
Purification steps:
After reduction, dextran, N- and O-glycans were desalted with cation exchange SPE followed by graphitic carbon SPE.
Sample name:
N-linked oligosaccharides
N/A
Instrument manufacturer:
Thermo Fisher
Instrument model:
Scientific Velos Plus
Customizations:
N/A
Ion mode:
Negative
Software name:
Xcalibur
Version:
3.0
Upgrades not reflected in version number:
N/A
Switching criteria (tandem only):
DDA, Top 5
Isolation width (global, or by MS level):
2
Location of ‘parameters’ file:
S-1 PGC LC ESI MSMS Methods (PDF)
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:
Octopole
Collision-Induced Dissociation (CID)
Gas composition:
Helium
Gas pressure:
N/A
Collision energy CID/function:
33% Normalized collision energy
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:
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:
Electron multiplier
UniCarb-DR 
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