Materials and Reagents

1-week-old Arabidopsis T-87 cells (50 g fresh weight)
Bio-Rad Protein Assay (Bio-Rad Laboratories, catalog number: 5000006JA )
2 mM synthesized substrate peptide [e.g., (OAOSOT)₃S] [using standard Fmoc solid-phase synthesis chemistry on a 431A peptide synthesizer (Life Technologies)]
2 mM Uridine 5’-diphosphogalactose disodium salt (Sigma-Aldrich, catalog number: U4500 )
1 M MOPS-KOH (pH 7.0)
10 mM MnCl₂
10% TX-100
1% Formic acid
Acetonitrile (HPLC grade) containing 0.1% formic acid
Water (HPLC grade) containing 0.1% formic acid
Tris-HCl (pH 7.0)
MgCl₂
Dithiothreitol
Leupeptin
Phenylmethanesulfonyl fluoride
Sucrose
Extraction buffer (see Recipes)
Suspension buffer (see Recipes)

Equipment

Waring blender
Miracloth (Merck Millipore Corporation, catalog number: 475855 )
Ultracentrifuge
30 °C incubator
Micro centrifuge
Semi-micro HPLC system (JASCO International Co., model: Micro21LC )
LCQ Deca XP-plus ESI ion-trap mass spectrometer (Thermo Fisher Scientific)
TSK-gel Amide-80 (3 μm) column (2 x 150 mm) (Tosoh Bioscience LLC, catalog number: 21865 )

Procedure

Preparation of Arabidopsis microsomal membranes
1. Arabidopsis T-87 cells are maintained on a 1-week culture interval under continuous darkness at 22 °C with shaking at 120 rpm.
2. Suspend 1-week-old Arabidopsis T-87 cell (50 g fresh weight) in 20 ml Extraction buffer.
3. Cool off to 4 °C on ice.
4. Homogenize at 20,000 rpm for 5 min at 4 °C in a Waring blender.
5. Cool off to 4 °C on ice.
6. Filter the slurry through two layers of Miracloth.
7. Centrifuge the filtrate at 3,000 x g for 15 min at 4 °C.
8. Centrifuge the supernatant at 100,000 x g for 30 min at 4 °C.
9. Suspend the pellet (microsomal membranes: approximately 150 μg/μl) in 500 μl Suspension buffer by gentle pipetting.
10. Determine the protein concentration by conventional Bradford assay according to the manufacturer’s protocol (Bio-Rad Protein Assay).

Hyp O-galactosyltransferase activity assay

Set up 20 μl HPGT assay reactions in 0.5 ml microcentrifuge tube as follows.

HPGT assay components	Amount per reaction
1 M MOPS-KOH (pH 7.0)	2 μl
2 mM UDP-α-D-galactose	5 μl
10 mM MnCl₂	2 μl
10% TX-100	2 μl
2 mM substrate peptide	1 μl
Arabidopsis T-87 microsomal membranes	30 μg protein equivalent
WaterTotal	20 μl

Incubate at 30 °C, 1 h.
Add 2 μl 1% formic acid to stop reaction.
Add 80 μl acetonitrile.
Centrifuge at 20,000 x g for 5 min.

LC/MS analysis
10 μl aliquots of the assay solution were analyzed by LC-MS using a micro HPLC (high pressure liquid chromatography) system connected to an LCQ Deca XP-plus ESI ion-trap mass spectrometer. Chromatographic separation is performed by normal-phase HPLC on a TSK-gel Amide-80 (3 μm) column (2 x 150 mm).
1. The mobile phase is composed of HPLC grade water containing 0.1% formic acid (eluent A) and HPLC grade acetonitrile containing 0.1% formic acid (eluent B). The column temperature is maintained at 25 °C.
2. The HPLC flow rate is 100 μl/min, and the elution gradient was 60 to 30% B over 15 min.
3. The HPLC eluate was introduced into an electrospray ionization (ESI) ion-trap mass spectrometer in the positive ionization mode.
4. MS source parameters are as follows [e.g., (OAOSOT)₃S peptide]:
  1. Capillary temperature: 200 °C
  2. Capillary voltage: 42 V
  3. Source voltage: 5 kV
  4. Source current: 8.5 μA
  5. Sheath gas flow: 50
  6. Aux gas flow: 0
  7. Sweep gas flow: 0
  8. The mass range: m/z 500-2,000
5. The mass spectra are obtained by selected ion monitoring. [e.g., (OAOSOT)₃S: m/z 951.1, Galactosylated product: m/z 1032.2] (Figure 2).
  
  Figure 2. Selected ion chromatogram of substrate peptide and the galactosylated product. Substrate peptide was incubated with solubilized membrane fractions in the presence of UDP-α-D-galactose, then analyzed by LC-MS with selected ion monitoring of the substrate (m/z 951.1) and the galactosylated product (m/z 1032.2).

Recipes

Extraction buffer (prepare freshly and keep on ice)
25 mM Tris-HCl (pH 7.0)
10 mM MgCl₂
2 mM dithiothreitol
2 μM leupeptin
2 mM phenylmethanesulfonyl fluoride
250 mM sucrose
Suspension buffer
10 mM Tris-HCl (pH 7.0)
250 mM sucrose

Acknowledgments

This is the detailed protocol for the detection of HPGT activity described by Ogawa-Ohnishi and Matsubayashi (2015). This research was supported by a Grant-in-Aid for Scientific Research (S) from the Ministry of Education, Culture, Sports, Science, and Technology (No. 25221105).

References

Ogawa-Ohnishi, M. and Matsubayashi, Y. (2015). Identification of three potent hydroxyproline O-galactosyltransferases in Arabidopsis. Plant J 81(5): 736-746.
Ogawa-Ohnishi, M., Matsushita, W. and Matsubayashi, Y. (2013). Identification of three hydroxyproline O-arabinosyltransferases in Arabidopsis thaliana. Nat Chem Biol 9(11): 726-730.