Materials and Reagents

Cotton
1 ml Micropipette tip
Hyp O-galactosylated peptides or proteins
0.22 M Ba(OH)₂
0.32 M sulfuric acid
1 M NaOH
1 M HCl
10% aqueous ammonia
80% acetonitrile containing 0.1% formic acid
99.9% acetonitrile (HPLC grade) containing 0.1% formic acid
Water (HPLC grade) containing 0.1% formic acid

Equipment

Heat block
Centrifugal evaporator
BT AG 50W-X8 Resin (100-200 mg resin, H+ form) (Bio-Rad Laboratories, catalog number: 143-5441 )
Micro centrifuge
Micro HPLC system (JASCO International Co., model: micro21 LC-01 )
LCQ Deca XP-plus ESI ion-trap mass spectrometer (Thermo Fisher Scientific)
TSK-gel Amide-80 (3 μm) column (2.0 x 150 mm) (Tosoh Bioscience LLC, catalog number: 21865 )

Procedure

Ba(OH)₂ hydrolysis
1. Dissolve galactosylated peptide in 500 μl 0.22 M Ba(OH)₂ in a glass vial with cap.
2. Incubate at 105 °C, 6 h.
3. Incubate on ice for 5 min.
4. Add 500 μl of 0.32 M sulfuric acid on ice.
5. Centrifuge at 20,000 x g for 5 min.
Partial purification of Ba(OH)₂ hydrolysate (Figure 1)
1. Plug a 1 ml micropipette tip with a small amount of cotton.
2. Pack 200 mg AG 50W-X8 resin into the tip column.
3. Wash the column with 1 ml of 1 M NaOH by gravity flow.
4. Wash the column with 1 ml of 1 M HCl by gravity flow.
5. Wash the column with 1 ml of water by gravity flow.
6. Apply supernatant of the Ba(OH)₂ hydrolysate of the galactosylated peptide to the tip column.
7. Wash the column with 1 ml of water by gravity flow.
8. Elute with 1 ml of 10% aqueous ammonia by gravity flow.
9. Evaporate the sample to dryness.
10. Dissolve in 100 μl 80% acetonitrile containing 0.1% formic acid.
  
  Figure 1. Partial purification of Ba(OH)₂ hydrolysate. Supernatant of the Ba(OH)₂ hydrolysate of the galactosylated peptide was applied to the tip column.
LC/MS analysis
10 μl aliquots of the assay solution will be 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 40% B over 10 min.
3. Subject the HPLC eluate to coupled electrospray ionization (ESI) in the positive ionization mode.
4. MS source parameters are as follows:
  1. Capillary temperature: 200 V
  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 500-2000
    
    Figure 2. Detection of Hyp O-galactoside in Ba(OH)₂ hydrolysates of in vitro galactosylated AGP14 by LC-MS. The sample was analyzed by selected ion monitoring of Hyp (m/z 132.1) and Gal-Hyp (m/z 294.1). Ba(OH)₂ hydrolysis yields a diastereomeric pair of amino acids.
5. The mass spectra are obtained by selected ion monitoring in zoom scan mode (Hyp: m/z 132.1, Gal-Hyp: m/z 294.1).

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.