Methods related to glycan biochemistry

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2.1.22 Puridylamination (PA)-labelling of xyloglucans for analysis by HPLC

This method is based on (Kondo et al., 1990) and was used for labelling of xyloglucan in order to allow analysis of fucosidase activity assays or purification of xyloglucan substrate by HPLC. Reagent 1 and 2 were prepared at first. Reagent 1 contained 550 mg PA (2-aminopyridine, Sigma-Aldrich) resuspended in 200 µL of acetic acid. Solubilisation was triggered by heating up to maximally 80 °C. Reagent 2 was composed of 39 mg Borane-dimethylamine complex 97 % (Sigma-Aldrich) in 200 µL of acetic acid.

For labelling, 50 µL of reagent 1 were added to the dried oligosaccharide in a 1.5 mL Eppendorf tube. This mix was incubated at 90 °C for 90 minutes. The reactions were cooled to room temperature and then mixed with 50 µL of reagent 2 followed by incubation at 80 °C for 50 minutes. The reactions were cooled at room temperature and mixed with 500 µL of acetone. The reactions were intensively mixed and then centrifuged at 13 000 rpm for 5 minutes. The supernatant was discarded and the pellet was washed three times with acetone. Washing was continued until the pellet lost its yellow colour. The pellet was then dried and resuspended in 200 µL of water before HPLC-analysis or purification.

2.1.23 Separation of xyloglucans by HPLC

HPLC was performed using 50 mM ammonium-acetate buffer at pH 4 as buffer 1 and 25 % acetonitrile in buffer 1 as buffer 2. Separation was done by using a reversed phase column (Thermo Quest Hypersil Division, 250x4 mm, 5 µ Hypersil) at a flow rate of 1 mL per minute. The programme is given in Table 21and the flow rate was 1 mL per minute. Detection was performed with a fluorescence detector at 320 / 400 nm.

min % buffer B 0 10

25 45.7 26 10 30 10

Table 21: Overview of the HPLC gradient program used for separation of xyloglucans.  

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2.1.24 Purification of substrate XXFG-PA by HPLC and verification by MS

XXFG-PA was purified by HPLC as described above. All collected fractions were dried in a Speed-Vac and resuspended in 5 µL of deionised water. 0.7 µL of 1:2, 1:4 and 1:8 diluted samples were spotted on a MALDI-plate and vacuum-dried. The samples were covered with DHB (2,3-Dihydroxybenzoic acid, Fluka) and dried again.

Analysis has been performed by MALDI-TOF-MS.

2.1.25 Purification and digestion of RG-II

RG-II were purified by isolating the alcohol insoluble residue (AIR). Up to 500 mg of leaves or seedlings were smashed in 2 mL of 80 % ethanol in a mortar. The solution was transferred into a 2 mL Eppendorf tube and centrifuged at full speed for 3 minutes.

The pellet was then washed with 1 - 2 mL of 80 % ethanol, 95 % ethanol and finally acetone. The pellet was dried by SpeedVac without heat and then pulverised with a glass bar. Digestion of this pulverised cell wall powder followed with either rapidase in order to release the full RG-II molecule or TFA in order to release the side chains. 

2.1.25.1 Enzymatic release of whole RG-II monomers

The pulverised cell wall powder was suspended in McIlvaine buffer pH 5 (10 % w/v) complemented with Rapidase Vegetable Juice solution (1 % v/v) kindly given by DSM Food Specialities, Seclin, France. The reaction was performed at 37 °C for 24 hours.

2.1.25.2 Release of side chains by mild TFA-hydrolysis

For analysis, 5 mg of cell wall powder were mixed with 300 to 500 µL of 0.1 M TFA (Trifluoroacetic acid, Sigma-Aldrich) and incubated at 80 °C for 1 hour with occasional vortexing. The reaction mix was centrifuged at full speed for a few minutes and supernatant was collected. Before reduction, the reaction mix was basified with 1:10 diluted pure ammonia (Roth) to maximally pH 9.0.

2.1.25.3 Reduction of released RG-II

Reduction of released RG-II or side chains was performed using sodium borohydride (NaBH4, Roth). After basification with diluted ammoniac (NH3), the reaction mix was immediately reduced with 1 % (w/v) of NaBH4. The reaction was incubated for 3 hours at room temperature and immediately purified via carbon columns.

2.1.26 Purification of polysaccharides via carbon columns

Chemically or enzymatically released oligosaccharides from xyloglucans and RG-II were purified via carbon columns (Hypersep Hypercarb, Thermo Scientific). For small scale-preparations, one carbon column was washed with 700 µL of elution buffer (50 % of 0.3 % formic acid at pH 9.0 (adjusted with ammonium), 50 % of acetonitrile), followed by 700 µL of water and centrifugations at 1 000 rpm for 1 minute. The sample was then loaded and centrifuged. The column was washed again with 700 µL of water followed by elution with 350 µL of buffer elution buffer. The eluate was dried by speed vac and analysed by MALDI- or ESI-TOF-MS.

2.1.27 Analysis of xyloglucans by MALDI-TOF-MS

An aliquot of 0.7 µL of sample in 3 dilutions was spotted on a MALDI plate and dried in a vacuum. The dried spot was covered with 0.7 µL of 2 % [w/v] DHB (2,5-dihydroxy- benzoic acid) in 50 % acetonitrile and dried in a vacuum. Analysis has been performed on an Ultraflex II MALDI-TOF/TOF instrument (Bruker, Bremen, Germany) in positive reflectron mode.

2.1.28 Analysis of RG-II by ESI-TOF-MS

Chromatography has been performed using Hypercarb PGC columns (0.32  100 mm, GE Healthcare) online coupled to an ESI-Q-TOF Instrument (Micromas, Global Ultima). Starting buffer for chromatography of released chain A was 0.3 % formic acid brought to pH 3.0 with ammonia. A gradient over 20 minutes from 5 to 20 % acetonitrile (AcCN) was developed at a flow rate of 8 µL/min. Flow rate was maintained with an Ultimate 3000 (Dionex). Detection was made by positive mode ESI-MS with a capillary voltage of 3,2 kV, a cone voltage of 80 V, a gas flow of 75/150 l/hr, a source

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temperature of 100° and a cone temperature of 120°C. MS/MS experiments were conducted with 30 % dissociation energy and argon.

Full RG-II was analyzed using a trinär gradient, starting with deionised water containing 0.1% formic acid and increasing the content of AcCN to 15 % within 10 minutes. After this washing step, full RG-II was eluted switching to a formate puffer pH 9 (0.3% formic acid, brought to pH 9 with ammonia) and increasing the % of AcCN over 20 minutes to 40%.

ESI-MS analysis was performed with a capillary voltage of -2.7 kV and for MS/MS experiments 75 % dissociation energy was fixed.

Identification and quantification was obtained using MassLynx SP 4 software or in case of series of measurements using the automatic data mining software MassMap.