Swift Universal Glycan Acquisition (SUGA) Enables Quantitative Glycan Profiling Across Diverse Sample Types

Announcement date

2024/06/09

Responsible:

Richard D Cummings . Glycomics Core, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA

Description

The ability to rapidly analyze complex mixtures of glycans derived from glycoproteins is important, but the techniques are often laborious and require multiple glycan derivatization steps. Here we describe an approach termed Swift Universal Glycan Acquisition (SUGA) in which the total released, native, non-reduced N-glycan samples are analyzed following direct injection and electrospray ionization in a mass spectrometer with a rapid 3-minute run time for each sample. As electrospray ionization (ESI) can generate multiple adducts for the same glycan composition (MS1), deconvolution was performed to yield the relative intensity profile for each glycan composition detected; each annotated composition is supported by an annotated MS2 spectrum. This combination of MS1 and MS2 data enables confident glycan identification. The data obtained by SUGA were comparable to those obtained using permethylated N-glycans analyzed by matrix-assisted laser-desorption/ionization (MALDI)-MS. The SUGA approach was applied to the analyses of several purified glycoproteins and N-glycans derived from cells and compared to spectra obtained following permethylation and analysis by MALDI-MS.

Sample preparation

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1. Sample Origin

General information:
Novel data acquisition method involving glycan release, optional permethylation, clean-up, and acquisition by MS.

1.1 Biologically derived material

Biologically derived material - Recombinantly produced material

Cell type:
Expi-CHO

Growth/harvest conditions for recombinantly produced material:
ExpiCHO-S cells were cultured in ExpiCHO Expression medium (Thermo Fisher, Cat. A2910001) in a humidified 5% CO2 incubator at 37 °C and 100 rpm. Cells were maintained in a T25 flask until reaching log phase growth (4e6/mL). One day before drug treatment, cells were transferred to 6-well plates, 1e5 cells per well for a total volume of 2 mL.

Biologically derived material - Biological origin of Material

Origin (biological fluid, tissue, etc):
N/A

Species:
N/A

Treatments and/or storage conditions:
Glycosylation inhibitors were added to the culture medium at 5 µg/mL in triplicate for each time point. Every 24 hours for 4 days, each well was collected and cells collected by centrifugation and washed 3x with PBS yielding a cell pellet. Cell counts and viability were monitored upon collection, with each treatment having similar final cell counts (~2.2e6/mL). All treated cells were at greater than 90% viability after the full course of treatment.

Glycoprotein:
Q58D62

Biologically derived material - Purchased from commercial manufacturer

Vendor and applicable item information:
Purified glycoproteins were purchased from Sigma Aldrich.

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:
NEB

Sample material treated in-solution or immobilized? State also temperature, duration, volume, enzyme concentration:
60 µL of PNGase-F digestion solution was added (New England Biolabs, 1 unit, H2O) and incubated at 37 °C for 18 hours.

Chemical treatments

Define the technique for oligosaccharide release or other chemical modifications:
N/A

Reaction conditions (temperature, duration, volume and chemical concentrations):
N/A

2.2 Sample Processing - Modification

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:
Permethylation

Explain the type of modification employed:
Methylation of hydroxyl groups

Source of materials, description of kits used, reaction conditions and detailed workflow:
Iodomethane treatment in NaOH DMSO

2.3 Sample Processing - Purification

Purification steps:
Carbon clean was performed as per manufacturer’s protocol (Hypercarb 25 mg x 96 well plate, Thermo Fisher), which includes loading glycan (0.1% TFA), washing out salts (0.1% TFA), eluting the glycans (50% acetonitrile, 0.1% TFA), and drying down in a 96 well plate.

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:
Fusion Lumos

Customizations:
N/A

Ion mode:
Positive and Negative

(b) Control and analysis software

Software name:
Xcalibur

Version:
4.5

Upgrades not reflected in version number:
N/A

Switching criteria (tandem only):
N/A

Isolation width (global, or by MS level):
N/A

Location of ‘parameters’ file:
N/A

2. Ion sources

(a) Electrospray Ionisation (ESI)

Supply type (static, or fed):
Autosampler

Interface name:
Dionex Ultimate 3000

Catalog number, vendor, and any modifications made to the standard specification:
N/A

Sprayer name:
Optamax NG HESI Source

Sprayer type, coating, manufacturer, model and catalog number (where available):
N/A

Relevant voltages where appropriate (tip, cone, acceleration):
N/A

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

(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:
N/A

(a) Post-source componentry - Collision cell

Collision-Induced Dissociation (CID)

Gas composition:
Nitrogen

Gas pressure:
N/A

Collision energy CID/function:
33% normalised 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

(b) Post-source componentry - TOF drift tube

Reflectron status (on, off, none):
N/A

(c) Post-source componentry - Ion trap

Final MS stage achieved:
MS2

(d) Post-source componentry - Ion mobility

Gas:
N/A

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