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Purification and Analysis of Free Oligosaccharides in Saccharomyces cerevisiae
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Purification and Analysis of Free Oligosaccharides in Saccharomyces cerevisiae

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Category
Biosynthesis & Metabolism
Protocol Name

Purification and Analysis of Free Oligosaccharides in Saccharomyces cerevisiae

Authors
Hirayama, Hiroto
Glycometabolome Team, Systems Glycobiology Research Group, Chemical Biology Department, RIKEN
KeyWords
Reagents

YPD (2 % peptone, 1 % yeast extract, and 2 % glucose)

20 mM Tris-HCl (pH8.0)

100 % ethanol (Wako Pure Chemical Industries, Ltd., Osaka, Japan)

100 % acetonitrile (HPLC grade, Wako Pure Chemical Industries, Ltd.)

AG1-X2 (200 – 400 mesh; acetate form; Bio-Rad Laboratories, Hercules, CA)

AG50-X8 (200 – 400 mesh; H+ form; Bio-Rad Laboratories)

2 - aminopyridine (special grade for fluorescence labeling; Wako Pure Chemical Industries, Ltd.)

Dimethylamine-Borane (Wako Pure Chemical Industries, Ltd.)

Phenol (Wako Pure Chemical Industries, Ltd)

Chloroform (Wako Pure Chemical Industries, Ltd.)

Instruments

InertSep GC column (150 mg/3 mL; GL Sciences Inc., Tokyo, Japan)

NH2P-50 4E column (4.6 × 250 mm; Showa Denko K. K., Tokyo, Japan)

Speed Vac concentrator (EYELA, Tokyo, Japan)

HPLC system (LaChrom Elite; Hitachi High-Tech Science Corporation, Tokyo, Japan)

Methods
1.

Extraction of free oligosaccharides from yeast cells

1) 

 Grow over night culture (5 mL scale).

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2) 

 Dilute saturated cells to OD600 = 0.5 with YPD, and grow to OD600 = 2.0 (3–4 h) at 30˚C (50 mL scale).

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3) 

 Harvest the cells and resuspend 100 OD600 unit cells in 500 μL of 20 mM Tris-HCl (pH 8.0).

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4) 

 Further add 500 μL of 100% EtOH.

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5) 

 Mix and centrifuge at top speed for 5 min at 4˚C.

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6) 

 Transfer the supernatant to new tube, then lyophilize by Speed Vac or equivalent.

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2.

Desalting and purification of free oligosaccharides

1) 

 Resuspend lyophilized sample in 500 μL of DW.

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2) 

 Apply samples onto AG1-X2 and AG50-X8 (resin volume, 500 μL each) and wash the column with 3 mL water, then collect flow-through.

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3) 

 Apply the flow-through onto an equilibrated InertSep GC column.

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4) 

 Wash the column with 3 mL water (×2 times).

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5) 

 Elute purified free oligosaccharides with 3 mL of 25 % acetonitrile.

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6) 

 Evaporate samples to dryness by Speed Vac or equivalent.

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3.

Fluorescent labeling and HPLC analysis of free oligosaccharides

1) 

 Pyridylamino labeling and size-fractionation HPLC analysis of free oligosaccharides are carried out as described in Fluorescent labeling of glycans and HPLC analysis” written by Shin-ichi Nakakita and Shunji Natsuka in glycoPOD.

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Notes

If significant amount of glucose oligomers (derived from β-1,6-glucan in the cellwall) are contaminated in samples, the oligomers can be removed by treatment with an endo-1,6-β-glucanase as described previously [Hirayama et al., (2010) JBC] . 

Initial amount

100 OD600 unit cells

Produced amount

Approximately 500 pmol of fOSs (Hex5-12HexNac2 forms glycans)

Figure & Legends

Figure & Legends

 

 

Fig. 1.  Formation/catabolic pathway of fOSs in yeast.

On the luminal side of the ER, misfolded glycoproteins are recognized by the ERAD and retrotranslocated from the ER to the cytosol

(1). In the cytosol, Peptide:N-glycanase (Png1) cleaves N-glycans from misfolded glycoproteins and form fOSs. Several misfolded glycoproteins traffic between ER and Golgi and further mannosylated by a Golgi-localized mannosyltransferase, Och1, before their degradation (2). fOSs are trimmed a sole cytosolic α-mannosidase, Ams1, giving rise to M5 form fOSs.

This figure was originally published by Glycobiology. 21 (10): 1341–1348. 2011 “Metabolism of fOSs is facilitated in the och1Δ mutant of Saccharomyces cerevisiae” Hirayama H. and Suzuki T. Oxford University press

 

 

Fig. 2. The elution profile of yeast fOSs by size-fractionation HPLC (Shodex Asahipak NH2P-50).

 

Hex5–Hex12 represents Hex5HexNAc2–Hex12HexNAc2, respectively. The arrowheads indicate the elution position of PA-isomaltooligosaccharides (PA-glucose oligomer) for elution standard. *, non-specific peak.

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