Sequencing of heparan sulfate

Announcement date

2021/02/20

Responsible:

Rebecca Miller . University of Copenhagen

Description

Chemically synthesis heparan sulfate structures MS/MS DTIMS (He)

Sample preparation

---

1. Sample Origin

General information:
Chemically synthesis heparan sulfate structures MS/MS DTIMS

1.1 Biologically derived material

Biologically derived material - Recombinantly produced material

Cell type:
N/A

Growth/harvest conditions for recombinantly produced material:
Chemically synthesised structures

Biologically derived material - Biological origin of Material

Origin (biological fluid, tissue, etc):
tissue

Species:
Mus musculus (Mouse)

Treatments and/or storage conditions:
Porcine mucosal heparin

Glycoprotein:
Glycosaminoglycans not proteins

Biologically derived material - Purchased from commercial manufacturer

Vendor and applicable item information:
Chemically synthesis material from labs; Ralf Schwörer, Olga V. Zubkova, Peter. C. Tyler, Yongmei Xu, Jian Liu, Pradeep Chopra, Geert-Jan Boons.

1.2 Chemically derived material

Synthesis steps or specify where the equivalent reaction protocol is available:
The levulinoyl (Lev) esters of the sequences were removed by treatment with hydrazine acetate, and subjected to O-sulfation using sulfur trioxide/pyridine complex in DMF, followed by Fmoc removal with triethylamine/dichloromethane (1/4, v/v). The methyl esters and acetates were saponified with H2O2 and LiOH in THF. Next, the azide groups of the compounds were reduced under Staudinger conditions using PMe3/THF (1.0 M), and the resulting amines were N-sulfated, employing a sulfur trioxide/pyridine complex in MeOH in the presence of Et3N and NaOH. The target structures were obtained by hydrogenation, over Pd/C in a mixture of tBuOH/H2O (1/1, v/v) to cleave the protecting group of the linker followed by further hydrogenation over Pd(OH)2/C to remove the benzyl ethers,

Description of starting material:
Synthetically derived materials

2. Sample Processing

2.1 Sample Processing - Isolation

Enzymatic treatments

Enzymes used for oligosaccharide removal or modification of starting material:
Biosynthetic-BIOSYNTHETIC (SEQUENCE)

Describe vendor or expression and purification procedure:
Lab expressed

Sample material treated in-solution or immobilized? State also temperature, duration, volume, enzyme concentration:
5 mU 2O-sulfotransferase in 100 mM PAPS, 50 mM MES, pH 7 at 30oC overnight

Chemical treatments

Define the technique for oligosaccharide release or other chemical modifications:
Biosynthetic-BIOSYNTHETIC (SEQUENCE)

Reaction conditions (temperature, duration, volume and chemical concentrations):
5 mU 2O-sulfotransferase in 100 mM PAPS, 50 mM MES, pH 7 at 30oC overnight

2.2 Sample Processing - Modification

Enzymatic modifications

Describe any treatments made to the isolated material:
Biosynthetic-BIOSYNTHETIC (SEQUENCE)

Enzyme concentration, supplier, biological source, incubation time and temperature:
5 mU 2O-sulfotransferase in 100 mM PAPS, 50 mM MES, pH 7 at 30oC overnight

If novel glycosidase was used, provide information indicating the origin (i.e. species) of the enzyme:
Sf9 cells

Chemical modifications

Describe any treatments made to the isolated material:
Biosynthetic-BIOSYNTHETIC (SEQUENCE)

Explain the type of modification employed:
Method above

Source of materials, description of kits used, reaction conditions and detailed workflow:
5 mU 2O-sulfotransferase in 100 mM PAPS, 50 mM MES, pH 7 at 30oC overnight

2.3 Sample Processing - Purification

Purification steps:
Gel filtration and strong anion exchange

3. Defined Sample

Sample name:
Glycosaminoglycans

Liquid chromatography

---

N/A

MS

---

1. General features

(a) Global descriptors

Instrument manufacturer:
Waters

Instrument model:
Synapt MS

Customizations:
DTIMS

Ion mode:
Negative

(b) Control and analysis software

Software name:
MassLynx

Version:
4.1

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):
Waters

Interface name:
Static

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

Sprayer name:
NanoLock Spray Ionisation Source

Sprayer type, coating, manufacturer, model and catalog number (where available):
Pd/Pt-coated borosilicate nanoelectrospray ionization (nESI) capillaries

Relevant voltages where appropriate (tip, cone, acceleration):
Capillary Voltage: 0.65kV, Cone Voltage 2, Source Temp 100oC,

Degree of prompt fragmentation evaluated:
N/A

Whether in-source dissociation performed:
N/A

Other parameters if discriminant for the experiment (such as nebulizing gas and pressure):
Trap gas flow, 2 ml/min; helium cell gas flow, 180 ml/min; ion mobility gas flow, 20 ml/min; trap bias, 2V; ion mobility wave velocity, 300 m/s; ion mobility wave height, 0 V.

(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

N/A