LC/MS-based Detection of Hydroxyproline O-galactosyltransferase Activity    

Materials and Reagents

1. 1-week-old Arabidopsis T-87 cells (50 g fresh weight)
   
2. Bio-Rad Protein Assay (Bio-Rad Laboratories, catalog number: 5000006JA )
   
3. 2 mM synthesized substrate peptide [e.g., (OAOSOT)₃S] [using standard Fmoc solid-phase synthesis chemistry on a 431A peptide synthesizer (Life Technologies)]
   
4. 2 mM Uridine 5’-diphosphogalactose disodium salt (Sigma-Aldrich, catalog number: U4500 )
   
5. 1 M MOPS-KOH (pH 7.0)
   
6. 10 mM MnCl₂
   
7. 10% TX-100
   
8. 1% Formic acid
   
9. Acetonitrile (HPLC grade) containing 0.1% formic acid
   
10. Water (HPLC grade) containing 0.1% formic acid
    
11. Tris-HCl (pH 7.0)
    
12. MgCl₂
    
13. Dithiothreitol
    
14. Leupeptin
    
15. Phenylmethanesulfonyl fluoride
    
16. Sucrose
    
17. Extraction buffer (see Recipes)
    
18. Suspension buffer (see Recipes)
    

Equipment

1. Waring blender
   
2. Miracloth (Merck Millipore Corporation, catalog number: 475855 )
   
3. Ultracentrifuge
   
4. 30 °C incubator
   
5. Micro centrifuge
   
6. Semi-micro HPLC system (JASCO International Co., model: Micro21LC )
   
7. LCQ Deca XP-plus ESI ion-trap mass spectrometer (Thermo Fisher Scientific)
   
8. TSK-gel Amide-80 (3 μm) column (2 x 150 mm) (Tosoh Bioscience LLC, catalog number: 21865 )
   

Procedure

1. 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).
   
   
2. Hyp O-galactosyltransferase activity assay
   
   1. 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
      Water Total	20 μl

      
      
   2. Incubate at 30 °C, 1 h.
      
   3. Add 2 μl 1% formic acid to stop reaction.
      
   4. Add 80 μl acetonitrile.
      
   5. Centrifuge at 20,000 x g for 5 min.
   
   
3. 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

1. 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
   
2. 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

1. Ogawa-Ohnishi, M. and Matsubayashi, Y. (2015). Identification of three potent hydroxyproline O-galactosyltransferases in Arabidopsis. Plant J 81(5): 736-746.
   
2. 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.