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Hydroxyproline (Hyp) O-galactosylation is a plant-specific post-translational modification found in extracellular glycoproteins such as arabinogalactan proteins (AGPs). Hyp O-galactosylation is mediated by Hyp O-galactosyltransferase (HPGT) that catalyzes the transfer of a D-galactopyranosyl residue to the hydroxyl group of Hyp residues of peptides from the sugar donor UDP-α-D-Gal. Here we describe an LC/MS-based method for the detection of Hyp O-galactoside.

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Detection of Hydroxyproline O-galactoside by LC/MS

Plant Science > Plant biochemistry > Carbohydrate
Authors: Mari Ogawa-Ohnishi
Mari Ogawa-OhnishiAffiliation: Division of Biological Science, Graduate School of Science, Nagoya University Chikusa, Nagoya, Japan
Bio-protocol author page: a2863
 and Yoshikatsu Matsubayashi
Yoshikatsu MatsubayashiAffiliation: Division of Biological Science, Graduate School of Science, Nagoya University Chikusa, Nagoya, Japan
For correspondence: matsu@bio.nagoya-u.ac.jp
Bio-protocol author page: a2864
Vol 6, Iss 2, 1/20/2016, 733 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.1710

[Abstract] Hydroxyproline (Hyp) O-galactosylation is a plant-specific post-translational modification found in extracellular glycoproteins such as arabinogalactan proteins (AGPs). Hyp O-galactosylation is mediated by Hyp O-galactosyltransferase (HPGT) that catalyzes the transfer of a D-galactopyranosyl residue to the hydroxyl group of Hyp residues of peptides from the sugar donor UDP-α-D-Gal. Here we describe an LC/MS-based method for the detection of Hyp O-galactoside.

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.


How to cite: Ogawa-Ohnishi, M. and Matsubayashi, Y. (2016). Detection of Hydroxyproline O-galactoside by LC/MS. Bio-protocol 6(2): e1710. DOI: 10.21769/BioProtoc.1710; Full Text



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