N-glycans and O-glycans released from purified mammalian glycoprotein standards and proteins from cell lysate

Responsible:

Christopher Ashwood . Macquarie University . chrashwood@gmail.com

Sample preparation

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1.Sample Origin

General information:
Reduced but otherwise native N-glycans and O-glycans released from purified mammalian glycoprotein standards and proteins from cell lysate

Growth/harvest conditions for recombinantly produced material:
The J774A.1 cell line (ATCC® TIB-67) was cultured in a T-75 flask with 10mL of Dulbecco’s Modified Eagle’s Medium supplemented with 10% (v/v) fetal bovine serum. 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, centrifuged (500g for 10 minutes) and then the supernatant was removed, for a total of three washes.

Treatments and/or storage conditions:
The cells were then lysed and protein precipitated using a chloroform:methanol:water extraction. The precipitated protein was removed, resolubilized in 4M urea and protein yield quantified with the Bradford Protein assay.

For commercial material, vendor and applicable item information:
Bovine fetuin, Sigma Aldrich (Sydney, Australia), product: F3385. Human IgG, Sigma Aldrich (Sydney, Australia), product: I4506

Generation of chemically derived material:
NA

2. Sample Processing

Purification steps:
Cation exchange desalting: 25uL packed volume of AG 50W X8 cation exchange resin in Millipore C18 tips. Resin activated with 3x 50uL 1M HCl, 3x 50uL MeOH, 3x 50uL H2O. Samples suspended in 10uL of ultrapure water and added to column. Glycans eluted from column with 3x 50uL H2O and eluate collected and dried. Removal of borate: Samples dried in centrifugal evaporator and washed/dried 2x with 100uL methanol. Graphitised carbon enrichment: 2.5mg of graphitised carbon added to Millipore C18 tips. Resin washed with 3x 50uL ACNR containing 0.1% (v/v) TFA then 3x 50uL ultrapure water containing 0.1% (v/v) TFA. Sample suspended in 10uL of ultrapure water containing 0.1% (v/v) TFA and added to column. Column washed with 3x 50uL ACNR containing 0.1% (v/v) TFA and then glycans released from resin with 2x 10uL 50% (v/v) ACNR:ultrapure water with 0.1% (v/v) TFA. Glycans then dried in centrifugal evaporator.

2. Defined Sample

Sample name:
Oligosaccharides

HPLC

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1. General settings

Instrument: Thermo Fisher 1100 Series

Manufacturer url: NA

Injector: NA

Injector settings: NA

2. Column

Manufacturer: Thermo Fisher

Model: Hypercarb

Type of Chromatography: Reversed-phase HPLC

Type of Material: Porous graphitised carbon

Column diameter: 0.18

Column length: 100

Particle size: 3

Manufacturer url: NA

3. Method run

Temperature: 50

Solvent a: 10mM Ammonium Bicarbonate in ultra-pure H2O

Solvent b: 10mM ammoinium bicarbonate in 45% (v/v) acetonitrile and 55% (v/v) ultra-pure H2O

Other solvent: NA

Flow rate: 4ul per min

Flow gradient: NA

Run time: NA

Phase: Reversed

MS

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1. General features

Date stamp: Mon Mar 05 UTC 2018

Instrument: Thermo Fisher Scientific LTQ Velos QTOF

Customizations from the manufacturer's specification:
NA

ion_mode: -

2. Ion sources

(a) Electrospray ionisation - ESI

Sprayer type : Fed

Interface name: Dionex 3000 Ultimate

Other data for the Interface: 5200.0356, Thermo Scientific, UltiMate 3000 Cap. Flow RSLC-Nano SYS

Sprayer name: HESI-II Source

Other data for the sprayer: Heated ESI, metal coating, Thermo Scientific, HESI-II, IQLAAEGABBFACNMAGY

Relevant voltages: Tip: 3.2kV, Skimmer: 0, Multipole 00 Offset: -2V, Lens 0: -0.9V, Multipole 0 Offset: 8.5V, Lens 1:11V, Gate Lens Offset: 90V, Multipole 1 Offset: 10.5V, Front Lens: 8V.

Degree of prompt fragmentation evaluated: No

In-source dissociation performed: No

Other parameters if discriminant for the experiment: Capillary temp: 275 degrees celcius, source heater temp: 45 degrees celcius, sheath gas flow: 7.6 units, sheath gas: nitrogen.

(b) MALDI

Plate composition (or type): NA

Matrix composition: NA

Deposition technique: NA

Relevant voltages: NA

Degree of prompt fragmentation evaluated: NA

PSD (or LID/ISD) summary: NA

Operation with or without delayed extraction: NA

Laser type: NA

Other laser related parameters: NA

url: NA

3. Ion transfer optics

Hardware options: Quadrupole and octopole

3. Post-source componentry

Collision-Induced Dissociation - CID

CID. Gas composition: 99.999% Helium

CID. Gas pressure: NA

CID. Collision energy: Normalised collision energy: 33, Activation Q: 0.25, Activation time: 10ms

Electron Transfer Dissociation - ETD

ETD. Reagent gas: NA

ETD. Pressure: NA

ETD. Reaction time: NA

ETD. Reagent atoms: NA

Electron Capture Dissociation - ECD

ECD. Emitter type: NA

ECD. Voltage: NA

ECD. Current: NA

TOF drift tube

TOF. Reflectron status: NA

Ion Trap

Ion trap. Final MS stage achieved: NA

Ion mobility

Ion mobility. Gas type : NA

Ion mobility. Pressure: NA

Ion mobility. Instrument parameters: NA

FT-ICR

Peak selection: NA

FT-ICR. Pulse: NA

FT-ICR. Width: NA

FT-ICR. IR: Decay time: NA

FT-ICR. IR: NA

FT-ICR. Other parameters: NA

Detectors

Detector type: NA