N-glycans from Mayaro virus

Announcement date

2021/04/09

Responsible:

Daniela Barretto Barbosa Trivella . Brazilian Center for Research in Energy and Materials, Campinas, São Paulo - Brazil

Description

We are reporting the LC-MS/MS characterization of N-glycans released from E1/E2 glycoproteins from MAYV (Mayaro virus)

Sample preparation

---

1. Sample Origin

General information:
N-glycans released from E1/E2 glycoproteins from MAYV strain growth in Vero CCL81 cells

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):
MAYV strain IQT 4235 (GenBank accession number MK070491.1) isolated from a symptomatic patient in the Peruvian Amazon and propagated in Vero CCL81 cells (ATCC)

Species:
N-glycans from Mayaro virus isolated from Homo sapiens and propagated in Vero cells (ATCC CCL-81)

Treatments and/or storage conditions:
Viral purification from supernatant by PEG precipitation followed by sucrose gradient centrifugation. Storage at -80 oC

Glycoprotein:
Q8QZ72

Biologically derived material - Purchased from commercial manufacturer

Vendor and applicable item information:
N/A

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:
Glycosidase-PNGase-F

Describe vendor or expression and purification procedure:
New England Biolabs

Sample material treated in-solution or immobilized? State also temperature, duration, volume, enzyme concentration:
in gel (SDS-PAGE) cleaveage of N-glycans; 50 mM sodium phosphate buffer pH 7.5, 16 h at 37°C, 50 U of PNGase F at 500.000 U/mL

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

Chemical modifications

Describe any treatments made to the isolated material:
Procainamide labelling

Explain the type of modification employed:
PNGase-F Released N-glycans react with procainamide in a reductive amination

Source of materials, description of kits used, reaction conditions and detailed workflow:
10 ul of labelling solution [DMSO and glacial acetic acid (70/30), 110 mg/ml of procainamide and 60 mg/ml sodium cyanoborohydride] was incubated with 10 ul of N-glycans solution at 65o.C overnight. After incubation, mixture was dried under vacuum and resuspended in 10 uL of water for UPLC-MS/MS analyses.

2.3 Sample Processing - Purification

Purification steps:
N/A

3. Defined Sample

Sample name:
MAYV_N-glycans

Liquid chromatography

---

N/A

MS

---

1. General features

(a) Global descriptors

Instrument manufacturer:
Bruker Daltonics

Instrument model:
Impact II

Customizations:
N/A

Ion mode:
Positive

(b) Control and analysis software

Software name:
microTOF Control

Version:
4.3

Upgrades not reflected in version number:
N/A

Switching criteria (tandem only):
MS2 triggered from the survey scan of the 3 most intense peaks > 50 in intensity

Isolation width (global, or by MS level):
5 pts

Location of ‘parameters’ file:
N/A

2. Ion sources

(a) Electrospray Ionisation (ESI)

Supply type (static, or fed):
Liquidy Chromatography

Interface name:
N/A

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

Sprayer name:
Electrospray

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

Relevant voltages where appropriate (tip, cone, acceleration):
End Plate Offset: 500 V; Capillarity: 4500 V

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):
Nebuling gas: N2 at 4 Bar; Dry gas: 10 l/min; Dry temp: 200 o.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:
QqTOF

(a) Post-source componentry - Collision cell

Collision-Induced Dissociation (CID)

Gas composition:
N2

Gas pressure:
N/A

Collision energy CID/function:
8 eV stepping mode basic: Collision RF 400 to 2000 Vpp; Transfer Time 30 to 120 us, timing 80 to 20%; MS/MS only: Collision Energy 100 to 250%; Timing 50 to 50%

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:
N/A

(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:
Multichannel plate