Isolation and identification of phosphorylated lysine peptides by retention time difference combining dimethyl labeling strategy

  • Yechen Hu
  • Yejing Weng
  • Bo Jiang
  • Xiao Li
  • Xiaodan Zhang
  • Baofeng Zhao
  • Qiong Wu
  • Zhen Liang
  • Lihua ZhangEmail author
  • Yukui Zhang


Protein phosphorylation plays essential roles in various biological procedures. Despite the well-established enrichment strategies for O-phosphoproteomics, the intrinsic acid lability of N–P phosphoramidate bond (phosphorylation of histidine, arginine and lysine) has impaired the progress of N-phosphoproteomics. Herein, we reported a retention time difference combining dimethyl labeling (ReDD) strategy for the isolation and identification of phosphorylated lysine (pLys) peptides. By such a method, pLys peptide could be isolated under 100000-fold interference of non-phosphorylated peptides. Furthermore, ReDD strategy was applied to map pLys sites from E. coli samples, leading to the identification of 11 pLys sites, among which K26p that originating from autonomous glycyl radical cofactor was validated both in mass spectrometry and HPLC co-elution experiments. Furthermore, 112 pLys sites from 100 proteins were identified in HeLa cells. All these results demonstrate that ReDD could provide a first glimpse into Lys phosphorylation, and could be an important step toward the global perspective on protein phosphorylation.


phosphorylated lysine retention time difference dimethyl labeling N-phosphorylation proteome LC-MS/MS 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.



This work was supported by the National Key Research and Development Program of China (2017YFA0505003, 2016YFA0501401), the National Natural Science Foundation of China (21505133, 21725506, 91543201), the CAS Key Project in Frontier Science (QYZDY-SSW-SLH017), and Innovation Program from DICP, Chinese Academy of Sciences (DICP TMSR201601).


  1. 1.
    Krall AS, Christofk HR. Nature, 2017, 546: 357–358CrossRefGoogle Scholar
  2. 2.
    Wang H, Nicolay BN, Chick JM, Gao X, Geng Y, Ren H, Gao H, Yang G, Williams JA, Suski JM, Keibler MA, Sicinska E, Gerdemann U, Haining WN, Roberts TM, Polyak K, Gygi SP, Dyson NJ, Sicinski P. Nature, 2017, 546: 426–430CrossRefGoogle Scholar
  3. 3.
    Oslund RC, Kee JM, Couvillon AD, Bhatia VN, Perlman DH, Muir TW. J Am Chem Soc, 2014, 136: 12899–12911CrossRefGoogle Scholar
  4. 4.
    Fuhs SR, Meisenhelder J, Aslanian A, Ma L, Zagorska A, Stankova M, Binnie A, Al-Obeidi F, Mauger J, Lemke G, Yates JR Iii, Hunter T. Cell, 2015, 162: 198–210CrossRefGoogle Scholar
  5. 5.
    Fuhrmann J, Clancy KW, Thompson PR. Chem Rev, 2015, 115: 5413–5461CrossRefGoogle Scholar
  6. 6.
    Hindupur SK, Colombi M, Fuhs SR, Matter MS, Guri Y, Adam K, Cornu M, Piscuoglio S, Ng CKY, Betz C, Liko D, Quagliata L, Moes S, Jenoe P, Terracciano LM, Heim MH, Hunter T, Hall MN. Nature, 2018, 555: 678–682CrossRefGoogle Scholar
  7. 7.
    Deluca M, Boyer P D, Peter J B, Moyer R W, Ebner K E, Kreil G, Hultquist D E. Biochem Z, 1963, 338: 512-525Google Scholar
  8. 8.
    Peter J B. J Biol Chem, 1963, 238: 1180–1182Google Scholar
  9. 9.
    Saito H. Chem Rev, 2001, 101: 2497–2510CrossRefGoogle Scholar
  10. 10.
    Potel CM, Lin MH, Heck AJR, Lemeer S. Nat Meth, 2018, 15: 187–190CrossRefGoogle Scholar
  11. 11.
    Adam K, Hunter T. Lab Invest, 2018, 98: 233–247CrossRefGoogle Scholar
  12. 12.
    Kee JM, Oslund RC, Perlman DH, Muir TW. Nat Chem Biol, 2013, 9: 416–421CrossRefGoogle Scholar
  13. 13.
    Kee JM, Villani B, Carpenter LR, Muir TW. J Am Chem Soc, 2010, 132: 14327–14329CrossRefGoogle Scholar
  14. 14.
    Trentini DB, Suskiewicz MJ, Heuck A, Kurzbauer R, Deszcz L, Mechtler K, Clausen T. Nature, 2016, 539: 48–53CrossRefGoogle Scholar
  15. 15.
    Suskiewicz MJ, Clausen T. Cell Chem Biol, 2016, 23: 888–890CrossRefGoogle Scholar
  16. 16.
    Fuhrmann J, Subramanian V, Kojetin DJ, Thompson PR. Cell Chem Biol, 2016, 23: 967–977CrossRefGoogle Scholar
  17. 17.
    Trentini DB, Fuhrmann J, Mechtler K, Clausen T. Mol Cell Proteomics, 2014, 13: 1953–1964CrossRefGoogle Scholar
  18. 18.
    Fuhrmann J, Schmidt A, Spiess S, Lehner A, Turgay K, Mechtler K, Charpentier E, Clausen T. Science, 2009, 324: 1323–1327CrossRefGoogle Scholar
  19. 19.
    Junker S, Maaß S, Otto A, Michalik S, Morgenroth F, Gerth U, Hecker M, Becher D. Mol Cell Proteom, 2018, 17: 335–348CrossRefGoogle Scholar
  20. 20.
    Ouyang H, Fu C, Fu S, Ji Z, Sun Y, Deng P, Zhao Y. Org Biomol Chem, 2016, 14: 1925–1929CrossRefGoogle Scholar
  21. 21.
    Fuhrmann J, Subramanian V, Thompson PR. Angew Chem, 2015, 127: 14928–14931CrossRefGoogle Scholar
  22. 22.
    Schmidt A, Trentini DB, Spiess S, Fuhrmann J, Ammerer G, Mechtler K, Clausen T. Mol Cell Proteom, 2014, 13: 537–550CrossRefGoogle Scholar
  23. 23.
    Chen CC, Bruegger BB, Kern CW, Lin YC, Halpern RM, Smith RA. Biochemistry, 1977, 16: 4852–4855CrossRefGoogle Scholar
  24. 24.
    Chen CC, Smith DL, Bruegger BB, Halpern RM, Smith RA. Biochemistry, 1974, 13: 3785–3789CrossRefGoogle Scholar
  25. 25.
    Zetterqvist Ö, Engström L. Biochim Biophys Acta, 1967, 141: 523–532CrossRefGoogle Scholar
  26. 26.
    Zetterqvist Ö. Biochim Biophys Acta, 1967, 141: 540–546CrossRefGoogle Scholar
  27. 27.
    Zetterqvist Ö. Biochim Biophys Acta, 1967, 141: 533–539CrossRefGoogle Scholar
  28. 28.
    Smith DL, Chen CC, Bruegger BB, Holtz SL, Halpern RM, Smith RA. Biochemistry, 1974, 13: 3780–3785CrossRefGoogle Scholar
  29. 29.
    Matthews HR. Pharm Ther, 1995, 67: 323–350CrossRefGoogle Scholar
  30. 30.
    Yokoi F. J Biochem, 2003, 133: 607–614CrossRefGoogle Scholar
  31. 31.
    Hiraishi H, Yokoi F, Kumon A. J Biochem, 1999, 126: 368–374CrossRefGoogle Scholar
  32. 32.
    Attwood PV. Biochim Biophys Acta, 2013, 1834: 470–478CrossRefGoogle Scholar
  33. 33.
    Wong C, Faiola B, Wu W, Kennelly PJ. Biochem J, 1993, 296: 293–296CrossRefGoogle Scholar
  34. 34.
    Zhong H, Xiao X, Zheng S, Zhang W, Ding M, Jiang H, Huang L, Kang J. Nat Commun, 2013, 4: 1656–1662CrossRefGoogle Scholar
  35. 35.
    Zhou H, Ye M, Dong J, Corradini E, Cristobal A, Heck AJR, Zou H, Mohammed S. Nat Protoc, 2013, 8: 461–480CrossRefGoogle Scholar
  36. 36.
    Attwood PV, Piggott MJ, Zu XL, Besant PG. Amino Acids, 2007, 32: 145–156CrossRefGoogle Scholar
  37. 37.
    Besant P, Attwood P, Piggott M. Curr Protein Pept Sc, 2009, 10: 536–550CrossRefGoogle Scholar
  38. 38.
    Bertran-Vicente J, Serwa RA, Schümann M, Schmieder P, Krause E, Hackenberger CPR. J Am Chem Soc, 2014, 136: 13622–13628CrossRefGoogle Scholar
  39. 39.
    Bertran-Vicente J, Schümann M, Schmieder P, Krause E, Hackenberger CPR. Org Biomol Chem, 2015, 13: 6839–6843CrossRefGoogle Scholar
  40. 40.
    Wei YF, Matthews HR. Anal Biochem, 1990, 190: 188–192CrossRefGoogle Scholar
  41. 41.
    Perlova TY, Goloborodko AA, Margolin Y, Pridatchenko ML, Tarasova IA, Gorshkov AV, Moskovets E, Ivanov AR, Gorshkov MV. Proteomics, 2010, 10: 3458–3468CrossRefGoogle Scholar
  42. 42.
    Hoffmann R, Segal M, Otvos Jr. L. Anal Chim Acta, 1997, 352: 327–333CrossRefGoogle Scholar
  43. 43.
    Batth TS, Francavilla C, Olsen JV. J Proteome Res, 2014, 13: 6176–6186CrossRefGoogle Scholar
  44. 44.
    Zhang Z, Tan M, Xie Z, Dai L, Chen Y, Zhao Y. Nat Chem Biol, 2011, 7: 58–63CrossRefGoogle Scholar
  45. 45.
    Tan M, Peng C, Anderson KA, Chhoy P, Xie Z, Dai L, Park J, Chen Y, Huang H, Zhang Y, Ro J, Wagner GR, Green MF, Madsen AS, Schmiesing J, Peterson BS, Xu G, Ilkayeva OR, Muehlbauer MJ, Braulke T, Mühlhausen C, Backos DS, Olsen CA, McGuire PJ, Pletcher SD, Lombard DB, Hirschey MD, Zhao Y. Cell Metabolism, 2014, 19: 605–617CrossRefGoogle Scholar
  46. 46.
    Tan M, Luo H, Lee S, Jin F, Yang JS, Montellier E, Buchou T, Cheng Z, Rousseaux S, Rajagopal N, Lu Z, Ye Z, Zhu Q, Wysocka J, Ye Y, Khochbin S, Ren B, Zhao Y. Cell, 2011, 146: 1016–1028CrossRefGoogle Scholar
  47. 47.
    Azevedo C, Saiardi A. Adv Biol Regulation, 2016, 60: 144–150CrossRefGoogle Scholar
  48. 48.
    Wyborn NR, Messenger SL, Henderson RA, Sawers G, Roberts RE, Attwood MM, Green J. Microbiology, 2002, 6: 1015–1026CrossRefGoogle Scholar
  49. 49.
    Wagner AFV, Schultz S, Bomke J, Pils T, Lehmann WD, Knappe J. Biochem Biophys Res Commun, 2001, 285: 456–462CrossRefGoogle Scholar
  50. 50.
    Azevedo C, Livermore T, Saiardi A. Mol Cell, 2015, 58: 71–82CrossRefGoogle Scholar

Copyright information

© Science China Press and Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Yechen Hu
    • 1
    • 2
  • Yejing Weng
    • 1
    • 2
  • Bo Jiang
    • 1
  • Xiao Li
    • 1
  • Xiaodan Zhang
    • 1
  • Baofeng Zhao
    • 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