MIRAGE report

The Glycomic MS Database and Repository

Announcement date

2025/10/19

Responsible:

Christopher Ashwood . Protea Glycosciences

Description

O-acetylation of sialic acids represents an additional layer of structural diversity and biological complexity, occurring at various hydroxyl positions (commonly C-7, C-8, or C-9) of the sialic acid residue. This modification modulates the recognition of sialylated glycans by lectins, antibodies, and viral proteins, and contributes to viral tropism and host susceptibility, particularly in influenza and coronaviruses that bind O-acetylated sialylated receptors. However, current LC-MS glycomics workflows commonly employ reduction or permethylation, which, while improving chromatographic stability and ionisation, result in the loss of labile O-acetyl groups, obscuring their biological relevance. Native glycan analysis, in contrast, preserves the complete structural integrity of glycans, enabling accurate detection of labile modifications. Using a native released glycan workflow limited to pH ≤ 8, O-acetylated N-glycans were detected in mouse and rat sera that were previously undetectable under basic derivatisation conditions. Beam-type collision-induced dissociation generated the most informative fragmentation spectra, with diagnostic ions confirming O-acetylated NeuGc and NeuAc residues. Chromatographic profiling revealed later elution and broadened peak shapes for O-acetylated species, consistent with increased hydrophobicity and microheterogeneity. A checkpoint-based identification workflow incorporating isotopic, chromatographic, and MS2 criteria reduced false positives, retaining only 3-5% of putative O-acetylated glycans as confident identifications. Quantitative comparison across species revealed extensive O-acetylation in rat (53.4%) and moderate modification in mouse (8.8%), but none detectable in human serum. These findings establish a robust analytical framework for native detection and characterisation of O-acetylated N-glycans, revealing species-specific regulation of this labile modification.

Sample preparation

1. Sample Origin

General information:
All chemicals and reagents were purchased from Millipore Sigma (Castle Hill, Australia) unless specified otherwise.

1.1 Biologically derived material

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):
N/A

Species:
Mus musculus (Mouse)

Treatments and/or storage conditions:
N/A

Glycoprotein:
N/A

Biologically derived material - Purchased from commercial manufacturer

Vendor and applicable item information:
Sera (human, rat, and mouse) were purchased from Millipore Sigma (Castle Hill, Australia).

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

Sample material treated in-solution or immobilized? State also temperature, duration, volume, enzyme concentration:
). Immobilised protein on the miniprep silica columns was resuspended in 160 µL of 50 mM TEAB (pH 8) and 50 units of PNGase-F (Promega, Wisconsin, USA) and left to react at 37 °C overnight

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:
N/A

Explain the type of modification employed:
N/A

Source of materials, description of kits used, reaction conditions and detailed workflow:
N/A

2.3 Sample Processing - Purification

Purification steps:
Following glycan release, glycans were eluted with 400 μL of 0.1% formic acid and then desalted using Supelclean ENVI-Carb SPE (100 mg). The desalting procedure involved conditioning with 400 μL acetonitrile/0.1% formic acid, equilibration with 1.5 mL water/0.1% formic acid, sample loading, desalting with 1.5 mL water/0.1% formic acid, and final elution with 400 μL 50:50 acetonitrile:water containing 0.1% formic acid. The desalted glycan solutions were then dried by centrifugal evaporation. Glycans were resuspended in Milli-Q H2O and then transferred into a 96 well PCR plate for injection. For reduced N-glycan analysis, glycans were dried by centrifugal evaporation after elution from silica columns, then reduced by 1 M NaBH4 in 50 mM KOH and incubated for 3 hours at 50 °C.

3. Defined Sample

Sample name:
N-linked oligosaccharides

Liquid chromatography

1. Equipment

Manufacturer:
Thermo Fisher

Model:
Vanquish

Instrument details:
Thermofisher Scientific Vanquish Horizon HPLC with MP35N fittings, PEEK tubing, and Vipers.

1.1 Column details & characteristics

Manufacturer:
Thermo Fisher

Model:
Hypercarb

Separation Mode:
Reversed phase

Column length:
100 mm

Inner diameter:
1 mm

Total volume (if relevant):
N/A

Stationary Phase:
PGC 3 micron pore size

Column heater:
Vanquish

Additional accessories:
N/A

1.2 Mobile phase

Description of solvent:
Both mobile phases contained 5 mM HFIP and 5 mM butylamine, with mobile phase A composed of water and mobile phase B composed of 100% acetone. LC separation was performed at 150 µL/min.

Description of constituents:
N/A

1.3 Properties of the chromatographic run (parameters that may change over run time)

Time:
30 min

Gradient:
Glycans were separated over a 30 min run, with 0-15% B over 23 min, 100% B held for 3 min, then 100% A for 4 min.

Flow rate:
150 ul/min

Temperature:
90 C

Sample injection procedure

Volume:
3-10

Buffer:
H2O

Inline/offline:
Inline

Direct/loop (full/partial loop):
Partial loop

Flush conditions:
Loop kept inline for gradient

Sample storage temperature:
4 oC

Sample loop material:
Stainless steel modified with MP35N

Type of sample vials used:
96 well plate

1.4 Pre- and post run processes

(a) Pre-run process

Type:
N/A

Substance, standards:
N/A

Time:
N/A

Flow rate:
N/A

Type of gradient mixer:
N/A

Post separation events:
N/A

Column regeneration:
N/A

Equipment used for detection:
MS

Detector type:
MS

Detection system:
MS

Equipment settings

Wavelength(s):
N/A

Gain:
N/A

Frequency that is being detected:
N/A

Sampling frequency (e.g. 10 Hz):
N/A

Timescale over which data was collected:
N/A

(b) Post-run process

Type:
N/A

Substance, standards:
N/A

Time:
N/A

Flow rate:
N/A

Type of gradient mixer:
N/A

Post separation events:
N/A

Column regeneration:
N/A

Equipment used for detection:
MS

Detector type:
MS

Detection system:
MS

Equipment settings

Wavelength(s):
N/A

Gain:
N/A

Frequency that is being detected:
N/A

Sampling frequency (e.g. 10 Hz):
N/A

Timescale over which data was collected:
N/A

1.5 Column outputs - fractions (if separation purpose is preparative)

Fraction name:
N/A

(a) Description of the procedure by which the fractions were collected

Start time:
N/A

End time:
N/A

Mode (fixed or peak directed):
N/A

(b) Description of the individual fractions

Time of collection:
N/A

Volume:
N/A

Charge:
N/A

Flow split:
N/A

LC-MALDI spotting:
N/A

1.6 Data annotation

Database name:
N/A

Database version:
N/A

Database name:
GlyCombo

Database version:
v1.2

Software name:
Skyline

Software version:
25.1

Software URL:
skyline.ms

Peak selection:
Informed based on isotopes and manual integration

Peak quantitation:
Subtraction from noise, trapezoidal intergration

Trace output:
Skyline Assays

2. Exoglycosidase treatment

Supplier name:
N/A

Exoglycosidase preparation (ref to sample prep):
N/A

Reaction time (ref to sample prep):
N/A

Control:
N/A

Protocol:
N/A

MS

1. General features

(a) Global descriptors

Instrument manufacturer:
Thermo Fisher

Instrument model:
IQ-X

Customizations:
N/A

Ion mode:
Negative

(b) Control and analysis software

Software name:
Xcalibur

Version:
4.2

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

Software name:
Tribrid Tune

Version:
N/A

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):
Fed

Interface name:
Optamax

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

Sprayer name:
N/A

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

Relevant voltages where appropriate (tip, cone, acceleration):
-3000 kV spray voltage

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):
Sheath gas = 30 Aux gas = 20 Transfer tube = 370 C

(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:
Helium

Gas pressure:
N/A

Collision energy CID/function:
38% NCE with Resonant 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

(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:
N/A