Advertisement

Cleavable hydrophobic derivatization strategy for enrichment and identification of phosphorylated lysine peptides

  • Yechen Hu
  • Yang Li
  • Hang Gao
  • Bo Jiang
  • Xiaodan Zhang
  • Xiao Li
  • Qiong Wu
  • Zhen Liang
  • Lihua ZhangEmail author
  • Yukui Zhang
Research Paper
Part of the following topical collections:
  1. New Insights into Analytical Science in China

Abstract

Because of structural flexibility and acid lability, the identification of phosphorylated lysine (pLys) peptides is a great challenge. We report here a cleavable hydrophobic derivatization (CHD) strategy for the enrichment and identification of pLys peptides. First, 2,5-dioxopyrrolidin-1-yl-3-(decyldisulfanyl)propanoate was synthesized to react with dephosphorylated lysine peptides, and then the derived peptides were captured by a C18 column, followed by cleavage of the hydrophobic chain, with the specific label left on the target peptides for further identification. By CHD, the enrichment of pLys peptides from interfering peptides (1:1000 mass ratio) was achieved. Furthermore, CHD was applied to screen the pLys targets from Escherichia coli lysates, and 39 pLys sites from 35 proteins were identified. Gene Ontology (GO) analysis showed that these proteins played vital roles in catabolism, metabolism, biogenesis, and biosynthetic processes. All these results demonstrate that CHD might pave the way for comprehensive profiling of the pLys proteome.

Keywords

Phosphorylated lysine peptides Cleavable hydrophobic derivatization N-Phosphorylation Proteomics Liquid chromatography–tandem mass spectrometry 

Notes

Acknowledgements

The authors are grateful for financial support from the National Key Research and Development Program of China (2017YFA0505003), the National Natural Science Foundation (91543201, 21505133, 21725506), the Chinese Academy of Sciences Key Project in Frontier Science (QYZDY-SSW-SLH017), and the Innovation Program of Dalian Institute of Chemical Physics, Chinese Academy of Sciences Key (DICP TMSR201601).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no competing interests.

Supplementary material

216_2019_1770_MOESM1_ESM.pdf (680 kb)
ESM 1 (PDF 679 kb)
216_2019_1770_MOESM2_ESM.xlsx (11 kb)
ESM 2 (XLSX 11 kb)

References

  1. 1.
    Murat S, Bigot M, Chapron J, Konig GM, Kostenis E, Battaglia G, et al. 5-HT2A receptor-dependent phosphorylation of mGlu2 receptor at serine 843 promotes mGlu2 receptor-operated Gi/o signaling. Mol Psychiatry. 2018.  https://doi.org/10.1038/s41380-018-0069-6.
  2. 2.
    Fuhs SR, Hunter T. pHisphorylation: the emergence of histidine phosphorylation as a reversible regulatory modification. Curr Opin Cell Biol. 2017;45:8–16.  https://doi.org/10.1016/j.ceb.2016.12.010.CrossRefGoogle Scholar
  3. 3.
    Besant P, Attwood P, Piggott M. Focus on phosphoarginine and phospholysine. Curr Protein Pept Sci. 2009;10(6):536–50.  https://doi.org/10.2174/138920309789630598.CrossRefGoogle Scholar
  4. 4.
    Riley NM, Coon JJ. Phosphoproteomics in the age of rapid and deep proteome profiling. Anal Chem. 2016;88(1):74–94.  https://doi.org/10.1021/acs.analchem.5b04123.CrossRefGoogle Scholar
  5. 5.
    Ciesa J, Fraczyk T, Rode W. Phosphorylation of basic amino acid residues in proteins: important but easily missed. Acta Biochim Pol. 2011;58:137–47.Google Scholar
  6. 6.
    Zetterqvist Ö, Engström L. Isolation of N-ε-[32P]phosphoryl-lysine from rat-liver cell sap after incubation with [32P]adenosine triphosphate. Biochim Biophys Acta. 1967;141(3):523–32.  https://doi.org/10.1016/0304-4165(67)90181-x.CrossRefGoogle Scholar
  7. 7.
    Zetterqvist Ö. Further studies on acid-labile [32P]phosphate bound to high-molecular weight material from rat-liver cell sap after incubation with [32P]adenosine triphosphate. Biochim Biophys Acta. 1967;141(3):533–9.  https://doi.org/10.1016/0304-4165(67)90182-1.CrossRefGoogle Scholar
  8. 8.
    Wålinder O, Zetterqvist Ö, Engström L. Purification of a bovine liver protein rapidly phosphorylated by adenosine triphosphate. Isolation of 1-32P-phosphohistidine 3-32P-phosphohistidine and N-ε-32P-phospholysine from 32P-labeled protein. J Biol Chem. 1968;243(10):2793–8.Google Scholar
  9. 9.
    Wålinder O. Identification of a phosphate-incorporating protein from bovine liver as nucleoside diphosphate kinase and isolation of 1-32P-phosphohistidine 3-32P-phosphohistidine and N-ε-32P-phospholysine from erythrocytic nucleoside diphosphate kinase incubated with adenosine triphosphate-32P. J Biol Chem. 1968;243(14):3947–52.Google Scholar
  10. 10.
    Wei Y, Matthews HR. Identification of phosphohistidine in proteins and purification of protein-histidine kinases. Methods Enzymol. 1991;200:388–414.  https://doi.org/10.1016/0076-6879(91)00156-Q.CrossRefGoogle Scholar
  11. 11.
    Ohmori H, Kuba M, Kumon A. Two phosphatases for 6-phospholysine and 3-phosphohistidine from rat brain. J Biol Chem. 1993;268:7625–7.Google Scholar
  12. 12.
    Hiraishi H, Yokoi F, Kumon A. 3-phosphohistidine and 6-phospholysine are substrates of a 56-kDa inorganic pyrophosphatase from bovine liver. Arch Biochem Biophys. 1998;349(2):381–7.  https://doi.org/10.1006/abbi.1997.0480.CrossRefGoogle Scholar
  13. 13.
    Hiraishi H, Yokoi F, Kumon A. Bovine liver phosphoamidase as a protein histidine/lysine phosphatase. J Biochem. 1999;126(2):368–74.  https://doi.org/10.1093/oxfordjournals.jbchem.a022459.CrossRefGoogle Scholar
  14. 14.
    Ek P, Ek B, Zetterqvist O. Phosphohistidine phosphatase 1 (PHPT1) also dephosphorylates phospholysine of chemically phosphorylated histone H1 and polylysine. Ups J Med Sci. 2015;120(1):20–7.  https://doi.org/10.3109/03009734.2014.996720.CrossRefGoogle Scholar
  15. 15.
    Chen CC, Smith DL, Bruegger BB, Halpern RM, Smith RA. Occurrence and distribution of acid-labile histone phosphates in regenerating rat liver. Biochem. 1974;13:3785–9.  https://doi.org/10.1021/bi00715a026.CrossRefGoogle Scholar
  16. 16.
    Chen CC, Bruegger BB, Kern CW, Lin YC, Halpern RM, Smith RA. Phosphorylation of nuclear proteins in rat regenerating liver. Biochem. 1977;16(22):4852–5.  https://doi.org/10.1021/bi00641a016.CrossRefGoogle Scholar
  17. 17.
    Bertran-Vicente J, Schümann M, Schmieder P, Krausea E, Hackenberger CPR. Direct access of site-specifically phosphorylated lysine peptides from solid-support. Org Biomol Chem. 2015;13:6839–43.  https://doi.org/10.1039/C5OB00734H.CrossRefGoogle Scholar
  18. 18.
    Bertran-Vicente J, Serwa RA, Schumann M, Schmieder P, Krause E, Hackenberger CP. Site-specifically phosphorylated lysine peptides. J Am Chem Soc. 2014;136(39):13622–8.  https://doi.org/10.1021/ja507886s.CrossRefGoogle Scholar
  19. 19.
    Bertran-Vicente J, Schumann M, Hackenberger CP, Krause E. Gas-phase rearrangement in lysine phosphorylated peptides during electron-transfer dissociation tandem mass spectrometry. Anal Chem. 2015;87(14):6990–4.  https://doi.org/10.1021/acs.analchem.5b01389.CrossRefGoogle Scholar
  20. 20.
    Boersema PJ, Raijmakers R, Lemeer S, Mohammed S, Heck AJ. Multiplex peptide stable isotope dimethyl labeling for quantitative proteomics. Nat Protoc. 2009;4(4):484–94.  https://doi.org/10.1038/nprot.2009.21.CrossRefGoogle Scholar
  21. 21.
    Lin S, Garcia BA. Examining histone posttranslational modification patterns by high-resolution mass spectrometry. Methods Enzymol. 2012;512:3–28.  https://doi.org/10.1016/B978-0-12-391940-3.00001-9.CrossRefGoogle Scholar
  22. 22.
    Huesgen PF, Lange PF, Rogers LD, Solis N, Eckhard U, Kleifeld O, et al. LysargiNase mirrors trypsin for protein C-terminal and methylation-site identification. Nat Methods. 2015;12(1):55–8.  https://doi.org/10.1038/nmeth.3177.CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Yechen Hu
    • 1
    • 2
  • Yang Li
    • 1
    • 2
  • Hang Gao
    • 1
    • 2
  • Bo Jiang
    • 1
  • Xiaodan Zhang
    • 1
  • Xiao Li
    • 1
  • Qiong Wu
    • 1
    • 2
  • Zhen Liang
    • 1
  • Lihua Zhang
    • 1
    Email author
  • Yukui Zhang
    • 1
  1. 1.CAS Key Laboratory of Separation Science for Analytical Chemistry, National Chromatographic R&A Center, Dalian Institute of Chemical PhysicsChinese Academy of SciencesDalianChina
  2. 2.University of Chinese Academy of SciencesBeijingChina

Personalised recommendations