Detection of Hydroxyproline O-galactoside by LC/MS   

Materials and Reagents

1. Cotton
   
2. 1 ml Micropipette tip
   
3. Hyp O-galactosylated peptides or proteins
   
4. 0.22 M Ba(OH)₂
   
5. 0.32 M sulfuric acid
   
6. 1 M NaOH
   
7. 1 M HCl
   
8. 10% aqueous ammonia
   
9. 80% acetonitrile containing 0.1% formic acid
   
10. 99.9% acetonitrile (HPLC grade) containing 0.1% formic acid
    
11. Water (HPLC grade) containing 0.1% formic acid
    

Equipment

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

Procedure

1. 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.
   
   
2. 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.
       
   
   
3. 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

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.