Discrimination of Isomers of Released N- and O-Glycans Using Diagnostic Product Ions in Negative Ion PGC-LC-ESI-MS/MS

Announcement date

2019/06/28

Responsible:

Nicolle H. Packer . Macquarie University, Australia

Description

We used porous graphitized carbon-LC-ESI-MS/MS to separate and detect released N- and O-glycan isomers from mammalian model glycoproteins using negative mode resonance activation CID-MS/MS. By interrogating similar fragment spectra from closely related glycan isomers that differ only in arm position and sialyl linkage, product fragment ions for discrimination between these features were discovered. These diagnostic ions were shown to be useful for isomer discrimination using both linear and 3D ion trap mass spectrometers when analyzing complex glycan mixtures from cell lysates. This platform-independent workflow can potentially be extended to automate the characterization and quantitation of other challenging glycan isomers.

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.

1.1 Biologically derived material

Biologically derived material - Recombinantly produced material

Cell type:
monocyte

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.

Biologically derived material - Biological origin of Material

Origin (biological fluid, tissue, etc):
Cell Line

Species:
Mus musculus (Mouse)

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.

Glycoprotein:
P12763

Biologically derived material - Purchased from commercial manufacturer

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

1.2 Chemically derived material

Synthesis steps or specify where the equivalent reaction protocol is available:
N/A

Description of starting material:
Glycoproteins

2. Sample Processing

2.1 Sample Processing - Isolation

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:
N-Glycans were released from PVDF membrane-bound protein using 1 U PNGase F with overnight incubation at 37°C

Chemical treatments

Define the technique for oligosaccharide release or other chemical modifications:
Labelling-REDUCTION

Reaction conditions (temperature, duration, volume and chemical concentrations):
Released N-glycans were reduced with 1 M NaBH4 in a 50- mM KOH solution for 3 h at 50°C, after which the reaction was neutralized by adding equimolar glacial acetic acid

2.2 Sample Processing - Modification

Enzymatic modifications

Describe any treatments made to the isolated material:
PNGase F

Enzyme concentration, supplier, biological source, incubation time and temperature:
Promega (Sydney, Australia)

If novel glycosidase was used, provide information indicating the origin (i.e. species) of the enzyme:
N-Glycans were released from PVDF membrane-bound protein using 1 U PNGase F with overnight incubation at 37°C

Chemical modifications

Describe any treatments made to the isolated material:
Labelling-REDUCTION

Explain the type of modification employed:
Reduction of reducing-end.

Source of materials, description of kits used, reaction conditions and detailed workflow:
Released N-glycans were reduced with 1 M NaBH4 in a 50- mM KOH solution for 3 h at 50°C, after which the reaction was neutralized by adding equimolar glacial acetic acid

2.3 Sample Processing - Purification

Purification steps:
Both Nglycans and O-glycans were desalted and enriched offline using AG 50W-X8 (Bio-Rad) strong cation exchange followed by PGC solid phase extraction micro-columns (Grace, Columbia, MD, USA).

3. Defined Sample

Sample name:
N-linked oligosaccharides

Liquid chromatography

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N/A

MS

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1. General features

(a) Global descriptors

Instrument manufacturer:
Thermo Fisher

Instrument model:
LTQ

Customizations:
N/A

Ion mode:
Negative

(b) Control and analysis software

Software name:
Xcalibur

Version:
3.1

Upgrades not reflected in version number:
N/A

Switching criteria (tandem only):
Top 5

Isolation width (global, or by MS level):
2 m/z

Location of ‘parameters’ file:
https://panoramaweb.org/GlycoMS2Diagnostics.url

Software name:
Compass for HCT/esquire

Version:
1.0

Upgrades not reflected in version number:
N/A

Switching criteria (tandem only):
Top 3

Isolation width (global, or by MS level):
4 m/z

Location of ‘parameters’ file:
https://panoramaweb.org/GlycoMS2Diagnostics.url

2. Ion sources

(a) Electrospray Ionisation (ESI)

Supply type (static, or fed):
Fed

Interface name:
Dionex 3000 Ultimate

Catalog number, vendor, and any modifications made to the standard specification:
5200.0356, Thermo Scientific, UltiMate 3000 Cap. Flow RSLC-Nano SYS

Sprayer name:
HESI-II Source

Sprayer type, coating, manufacturer, model and catalog number (where available):
Heated ESI, metal coating, Thermo Scientific, HESI-II, IQLAAEGABBFACNMAGY

Relevant voltages where appropriate (tip, cone, acceleration):
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

Whether in-source dissociation performed:
No

Other parameters if discriminant for the experiment (such as nebulizing gas and pressure):
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):
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

3. Ion transfer optics

Hardware options:
Quadrupole and octopole

(a) Post-source componentry - Collision cell

Collision-Induced Dissociation (CID)

Gas composition:
99.999% Helium

Gas pressure:
N/A

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

Electron Transfer Dissociation (ETD)

Reagent gas:
NA

Pressure:
NA

Reaction time:
NA

Number of reagent atoms:
NA

Electron Capture Dissociation (ECD)

Emitter type:
NA

Voltage:
NA

Current:
NA

(b) Post-source componentry - TOF drift tube

Reflectron status (on, off, none):
none

(c) Post-source componentry - Ion trap

Final MS stage achieved:
2

(d) Post-source componentry - Ion mobility

Gas:
NA

Pressure:
N/A

Instrument-specific parameters:
N/A

(e) Post-source componentry - FT-ICR

Peak selection:
N/A

Pulse:
N/A

Width:
N/A

Voltage:
N/A

Decay time:
N/A

IR:
N/A

Other parameters:
N/A

(f) Post-source componentry - Detectors

Detector type:
N/A