Analytical and Bioanalytical Chemistry

, Volume 407, Issue 22, pp 6637–6655 | Cite as

Structure characterization of unexpected covalent O-sulfonation and ion-pairing on an extremely hydrophilic peptide with CE-MS and FT-ICR-MS

  • Martin Pattky
  • Simone Nicolardi
  • Beatrix Santiago-Schübel
  • Daniel Sydes
  • Yuri E. M. van der Burgt
  • Antonia N. Klein
  • Nan Jiang
  • Jeannine Mohrlüder
  • Karen Hänel
  • Janine Kutzsche
  • S. A. Funke
  • D. Willbold
  • S. Willbold
  • C. HuhnEmail author
Paper in Forefront


In this study, we characterized unexpected side-products in a commercially synthesized peptide with the sequence RPRTRLHTHRNR. This so-called peptide D3 was selected by mirror phage display against low molecular weight amyloid-β-peptide (Aβ) associated with Alzheimer’s disease. Capillary electrophoresis (CE) was the method of choice for structure analysis because the extreme hydrophilicity of the peptide did not allow reversed-phase liquid chromatography (RPLC) and hydrophilic interaction stationary phases (HILIC). CE-MS analysis, applying a strongly acidic background electrolyte and different statically adsorbed capillary coatings, provided fast and efficient analysis and revealed that D3 unexpectedly showed strong ion-pairing with sulfuric acid. Moreover, covalent O-sulfonation at one or two threonine residues was identified as a result of a side reaction during peptide synthesis, and deamidation was found at either the asparagine residue or at the C-terminus. In total, more than 10 different species with different m/z values were observed. Tandem-MS analysis with collision induced dissociation (CID) using a CE-quadrupole-time-of-flight (QTOF) setup predominantly resulted in sulfate losses and did not yield any further characteristic fragment ions at high collision energies. Therefore, direct infusion Fourier transform ion cyclotron resonance (FT-ICR) MS was employed to identify the covalent modification and discriminate O-sulfonation from possible O-phosphorylation by using an accurate mass analysis. Electron transfer dissociation (ETD) was used for the identification of the threonine O-sulfation sites. In this work, it is shown that the combination of CE-MS and FT-ICR-MS with ETD fragmentation was essential for the full characterization of this extremely basic peptide with labile modifications.


Extremely hydrophilic peptide Capillary electrophoresis Coating Electron transfer dissociation Alzheimer’s disease 



The authors thank the Helmholtz Initiative and Networking Fund as well as the German Excellence Initiative commissioned by the German Research Foundation (DFG) for financial support.


  1. 1.
    Wimo A, Prince M (2010) World Alzheimer report 2010: the global economic impact of dementia. Alzheimer’s Disease International, London. Accessed 13 Jan 2015
  2. 2.
    van Groen T, Wiesehan K, Funke SA, Kadish I, Nagel-Steger L, Willbold D (2008) Reduction of Alzheimer’s disease amyloid plaque load in transgenic mice by D3, a D-enantiomeric peptide identified by mirror image phage display. Chem Med Chem 3:1848–1852CrossRefGoogle Scholar
  3. 3.
    Wiesehan K, Willbold D (2003) Mirror-image phage display: aiming at the mirror. Chem Bio Chem 4:811–815CrossRefGoogle Scholar
  4. 4.
    Funke SA, Willbold D (2009) Mirror image phage display—a method to generate d-peptide ligands for use in diagnostic or therapeutical applications. Mol BioSyst 5:783–786CrossRefGoogle Scholar
  5. 5.
    Sun N, Funke SA, Willbold D (2012) Mirror image phage display—generating stable therapeutically and diagnostically active peptides with biotechnological means. J Biotechnol 161:121–125CrossRefGoogle Scholar
  6. 6.
    van Groen T, Kadish I, Wiesehan K, Funke SA, Willbold D (2009) In vitro and in vivo staining characteristics of small, fluorescent, Aβ42-binding D-enantiomeric peptides in transgenic AD mouse models. Chem Med Chem 4:276–282CrossRefGoogle Scholar
  7. 7.
    Bartnik D, Funke SA, Andrei-Selmer L-C, Bacher M, Dodel R, Willbold D (2009) Differently selected D-enantiomeric peptides act on different Aβ species. Rejuvenation Res 13:202–205CrossRefGoogle Scholar
  8. 8.
    Olubiyi OO, Frenzel D, Bartnik D, Gluck JM, Brener O, Nagel-Steger L, Funke SA, Willbold D, Strodel B (2014) Amyloid aggregation inhibitory mechanism of arginine-rich D-peptides. Curr Med Chem 21:1448–1457CrossRefGoogle Scholar
  9. 9.
    Wang X, Carr PW (2007) An unexpected observation concerning the effect of anionic additives on the retention behavior of basic drugs and peptides in reversed-phase liquid chromatography. J Chromatogr A 1154:165–173CrossRefGoogle Scholar
  10. 10.
    van Groen T, Kadish I, Funke A, Bartnik D, Willbold D (2012) In: Rossen D (ed) Advances in protein chemistry and structural biology. Academic Press: Elsevier, AmsterdamGoogle Scholar
  11. 11.
    van Groen T, Kadish I, Funke SA, Bartnik D, Willbold D (2013) Treatment with D3 removes amyloid deposits, reduces inflammation, and improves cognition in aged AβPP/PS1 double transgenic mice. J Alzheimers Dis 34:609–620Google Scholar
  12. 12.
    Funke SA, van Groen T, Kadish I, Bartnik D, Nagel-Steger L, Brener O, Sehl T, Batra-Safferling R, Moriscot C, Schoehn G, Horn AHC, Müller-Schiffmann A, Korth C, Sticht H, Willbold D (2010) Oral treatment with the d-enantiomeric peptide D3 improves the pathology and behavior of Alzheimer’s disease transgenic mice. ACS Chem Neurosci 1:639–648CrossRefGoogle Scholar
  13. 13.
    Kohno T, Kusunoki H, Sato K, Wakamatsu K (1998) A new general method for the biosynthesis of stable isotope-enriched peptides using a decahistidine-tagged ubiquitin fusion system: an application to the production of mastoparan-X uniformly enriched with 15N and 15N/13C. J Biomol NMR 12:109–121CrossRefGoogle Scholar
  14. 14.
    Pattky M, Huhn C (2013) Advantages and limitations of a new cationic coating inducing a slow electroosmotic flow for CE-MS peptide analysis: a comparative study with commercial coatings. Anal Bioanal Chem 405:225–237CrossRefGoogle Scholar
  15. 15.
    Nicolardi S, Giera M, Kooijman P, Kraj A, Chervet J-P, Deelder A, van der Burgt YM (2013) On-line electrochemical reduction of disulfide bonds: improved FTICR-CID and -ETD coverage of oxytocin and hepcidin. J Am Soc Mass Spectrom 24:1980–1987CrossRefGoogle Scholar
  16. 16.
    Huhn C, Ramautar R, Wuhrer M, Somsen GW (2010) Relevance and use of capillary coatings in capillary electrophoresis-mass spectrometry. Anal Bioanal Chem 396:297–314CrossRefGoogle Scholar
  17. 17.
    Lam MY, Siu SO, Lau E, Mao X, Sun HZ, Chiu PN, Yeung WB, Cox D, Chu I (2010) Online coupling of reverse-phase and hydrophilic interaction liquid chromatography for protein and glycoprotein characterization. Anal Bioanal Chem 398:791–804CrossRefGoogle Scholar
  18. 18.
    McCalley DV (2005) Comparison of an organic polymeric column and a silica-based reversed-phase for the analysis of basic peptides by high-performance liquid chromatography. J Chromatogr A 1073:137–145CrossRefGoogle Scholar
  19. 19.
    Aitken A, Learmonth M (2003) In: Smith B (ed) Protein sequencing protocols, vol 211. Methods in molecular biology. Humana Press, New YorkGoogle Scholar
  20. 20.
    Medzihradszky KF, Darula Z, Perlson E, Fainzilber M, Chalkley RJ, Ball H, Greenbaum D, Bogyo M, Tyson DR, Bradshaw RA, Burlingame AL (2004) O-sulfonation of serine and threonine : mass spectrometric detection and characterization of a new post-translational modification in diverse proteins throughout the eukaryotes. Mol Cell Proteomics 3:429–440CrossRefGoogle Scholar
  21. 21.
    Shibue M, Mant CT, Hodges RS (2005) Effect of anionic ion-pairing reagent concentration (1–60 mM) on reversed-phase liquid chromatography elution behavior of peptides. J Chromatogr A 1080:58–67CrossRefGoogle Scholar
  22. 22.
    Lerro KA, Orlando R, Zhang HZ, Usherwood PNR, Nakanishi K (1993) Separation of the sticky peptides from membrane proteins by high-performance liquid chromatography in a normal-phase system. Anal Biochem 215:38–44CrossRefGoogle Scholar
  23. 23.
    Kostiainen R, Kauppila TJ (2009) Effect of eluent on the ionization process in liquid chromatography–mass spectrometry. J Chromatogr A 1216:685–699CrossRefGoogle Scholar
  24. 24.
    Neubert H, James I (2009) Online capillary weak cation exchange enrichment hyphenated to nanospray mass spectrometry for quantitation of a basic pegvisomant derived peptide. J Chromatogr A 1216:6151–6154CrossRefGoogle Scholar
  25. 25.
    Jandera P (2011) Stationary and mobile phases in hydrophilic interaction chromatography: a review. Anal Chim Acta 692:1–25CrossRefGoogle Scholar
  26. 26.
    Yoshida T (2004) Peptide separation by hydrophilic-interaction chromatography: a review. J Biochem Biophys Methods 60:265–280CrossRefGoogle Scholar
  27. 27.
    Zauner G, Deelder AM, Wuhrer M (2011) Recent advances in hydrophilic interaction liquid chromatography (HILIC) for structural glycomics. Electrophoresis 32:3456–3466CrossRefGoogle Scholar
  28. 28.
    Singer D, Kuhlmann J, Muschket M, Hoffmann R (2010) Separation of multiphosphorylated peptide isomers by hydrophilic interaction chromatography on an aminopropyl phase. Anal Chem 82:6409–6414CrossRefGoogle Scholar
  29. 29.
    Alpert AJ (2008) Electrostatic repulsion hydrophilic interaction chromatography for isocratic separation of charged solutes and selective isolation of phosphopeptides. Anal Chem 80:62–76CrossRefGoogle Scholar
  30. 30.
    Jiang W, Fischer G, Girmay Y, Irgum K (2006) Zwitterionic stationary phase with covalently bonded phosphorylcholine type polymer grafts and its applicability to separation of peptides in the hydrophilic interaction liquid chromatography mode. J Chromatogr A 1127:82–91CrossRefGoogle Scholar
  31. 31.
    Lämmerhofer M, Nogueira R, Lindner W (2011) Multi-modal applicability of a reversed-phase/weak-anion exchange material in reversed-phase, anion-exchange, ion-exclusion, hydrophilic interaction, and hydrophobic interaction chromatography modes. Anal Bioanal Chem 400:2517–2530CrossRefGoogle Scholar
  32. 32.
    Boysen RI, Yang Y, Chowdhury J, Matyska MT, Pesek JJ, Hearn MTW (2011) Simultaneous separation of hydrophobic and hydrophilic peptides with a silica hydride stationary phase using aqueous normal phase conditions. J Chromatogr A 1218:8021–8026CrossRefGoogle Scholar
  33. 33.
    Wilce MCJ, Aguilar M-I, Hearn MTW (1995) Physicochemical basis of amino acid hydrophobicity scales: evaluation of four new scales of amino acid hydrophobicity coefficients derived from RP-HPLC of peptides. Anal Chem 67:1210–1219CrossRefGoogle Scholar
  34. 34.
    Schmitt-Kopplin P, Frommberger M (2003) Capillary electrophoresis-mass spectrometry: 15 years of developments and applications. Electrophoresis 24:3837–3867CrossRefGoogle Scholar
  35. 35.
    Fonslow BR, Yates JR III (2009) Capillary electrophoresis applied to proteomic analysis. J Sep Sci 32:1175–1188CrossRefGoogle Scholar
  36. 36.
    Gennaro LA, Salas-Solano O (2009) Characterization of deamidated peptide variants by micropreparative capillary electrophoresis and mass spectrometry. J Chromatogr A 1216:4499–4503CrossRefGoogle Scholar
  37. 37.
    Gaus HJ, Beck-Sickinger AG, Bayer E (1993) Optimization of capillary electrophoresis of mixtures of basic peptides and comparison with HPLC. Anal Chem 65:1399–1405CrossRefGoogle Scholar
  38. 38.
    Timm V, Gruber P, Wasiliu M, Lindhofer H, Chelius D (2010) Identification and characterization of oxidation and deamidation sites in monoclonal rat/mouse hybrid antibodies. J Chromatogr B 878:777–784CrossRefGoogle Scholar
  39. 39.
    Lai M, Skanchy D, Stobaugh J, Topp E (1998) Capillary electrophoresis separation of an asparagine containing hexapeptide and its deamidation products. J Pharm Biomed Anal 18:421–427CrossRefGoogle Scholar
  40. 40.
    Timerbaev AR (2000) Element speciation analysis by capillary electrophoresis. Talanta 52:573–606CrossRefGoogle Scholar
  41. 41.
    Schug KA, Lindner W (2004) Noncovalent binding between guanidinium and anionic groups: focus on biological- and synthetic-based arginine/guanidinium interactions with phosph[on]ate and sulf[on]ate residues. Chem Rev 105:67–114CrossRefGoogle Scholar
  42. 42.
    Kim J, Zand R, Lubman DM (2002) Electrophoretic mobility for peptides with post-translational modifications in capillary electrophoresis. Electrophoresis 23:782–793CrossRefGoogle Scholar
  43. 43.
    Pattky M, Huhn C (2010) Protein glycosylation analysis with capillary-based electromigrative separation techniques. Bioanal Rev 2:115–155CrossRefGoogle Scholar
  44. 44.
    Young SY, Ye SH, Moo JS, Park J (1997) Analysis of phosphopeptides by capillary electrophoresis and matrix-assisted laser-desorption ionization-time-of-flight mass spectrometry. J Chromatogr A 763:285–293CrossRefGoogle Scholar
  45. 45.
    Dong Y-M, Chien K-Y, Chen J-T, Lin S-J, Yu J-S, Wang T-CV (2013) Site-specific separation and detection of phosphopeptide isomers with pH-mediated stacking capillary electrophoresis-electrospray ionization-tandem mass spectrometry. J Sep Sci 36:1582–1589CrossRefGoogle Scholar
  46. 46.
    Tadey T, Purdy WC (1995) Capillary electrophoretic resolution of phosphorylated peptide isomers using micellar solutions and coated capillaries. Electrophoresis 16:574–579CrossRefGoogle Scholar
  47. 47.
    Dawson JF, Boland MP, Holmes CFB (1994) A capillary electrophoresis-based assay for protein kinases and protein phosphatases using peptide substrates. Anal Biochem 220:340–345CrossRefGoogle Scholar
  48. 48.
    Boersema PJ, Mohammed S, Heck AJR (2009) Phosphopeptide fragmentation and analysis by mass spectrometry. J Mass Spectrom 44:861–878CrossRefGoogle Scholar
  49. 49.
    Seibert C, Sakmar TP (2008) Toward a framework for sulfoproteomics: synthesis and characterization of sulfotyrosine-containing peptides. Pept Sci 90:459–477CrossRefGoogle Scholar
  50. 50.
    Drake SK, Hortin GL (2010) Improved detection of intact tyrosine sulfate-containing peptides by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry in linear negative ion mode. Int J Biochem Cell Biol 42:174–179CrossRefGoogle Scholar
  51. 51.
    Kim J-S, Song S-U, Kim H-J (2011) Simultaneous identification of tyrosine phosphorylation and sulfation sites utilizing tyrosine-specific bromination. J Am Soc Mass Spectrom 22:1916–1925CrossRefGoogle Scholar
  52. 52.
    Li W, Backlund PS, Boykins RA, Wang G, Chen H-C (2003) Susceptibility of the hydroxyl groups in serine and threonine to β-elimination/Michael addition under commonly used moderately high-temperature conditions. Anal Biochem 323:94–102CrossRefGoogle Scholar
  53. 53.
    Yu Y, Hoffhines AJ, Moore KL, Leary JA (2007) Determination of the sites of tyrosine O-sulfation in peptides and proteins. Nat Methods 4:583–588CrossRefGoogle Scholar
  54. 54.
    Cantel S, Brunel L, Ohara K, Enjalbal C, Martinez J, Vasseur J-J, Smietana M (2012) An innovative strategy for sulfopeptides analysis using MALDI-TOF MS reflectron positive ion mode. Proteomics 12:2247–2257CrossRefGoogle Scholar
  55. 55.
    Stensballe A, Jensen ON, Olsen JV, Haselmann KF, Zubarev RA (2000) Electron capture dissociation of singly and multiply phosphorylated peptides. Rapid Commun Mass Spectrom 14:1793–1800CrossRefGoogle Scholar
  56. 56.
    Macek B, Mann M, Olsen JV (2009) Global and site-specific quantitative phosphoproteomics: principles and applications. Annu Rev Pharmacol Toxicol 49:199–221CrossRefGoogle Scholar
  57. 57.
    Monigatti F, Hekking B, Steen H (2006) Protein sulfation analysis—a primer. Biochim Biophys Acta Proteins Proteomics 1764:1904–1913CrossRefGoogle Scholar
  58. 58.
    Bakhtiar R, Guan Z (2006) Electron capture dissociation mass spectrometry in characterization of peptides and proteins. Biotechnol Lett 28:1047–1059CrossRefGoogle Scholar
  59. 59.
    Mikesh LM, Ueberheide B, Chi A, Coon JJ, Syka JEP, Shabanowitz J, Hunt DF (2006) The utility of ETD mass spectrometry in proteomic analysis. Biochim Biophys Acta Proteins Proteomics 1764:1811–1822CrossRefGoogle Scholar
  60. 60.
    Crowe MC, Brodbelt JS (2004) Infrared multiphoton dissociation (IRMPD) and collisionally activated dissociation of peptides in a quadrupole ion trap with selective IRMPD of phosphopeptides. J Am Soc Mass Spectrom 15:1581–1592CrossRefGoogle Scholar
  61. 61.
    Jaeger E, Remmer HA, Jung G, Metzger J, Oberthür W, Rücknagel KP, Schäfer W, Johann S, Zetl I (1993) Nebenreaktionen bei peptidsynthesen, V. O-sulfonierung von serin und threonin während der abspaltung der pmc- und mtr-schutzgruppen von argininresten bei fmoc-festphasen-synthesen. Biol Chem 374:349–362Google Scholar
  62. 62.
    Beck-Sickinger AG, Schnorrenberg G, Metzger J, Jung G (1991) Sulfonation of arginine residues as side reaction in Fmoc-peptide synthesis. Int J Pept Protein Res 38:25–31CrossRefGoogle Scholar
  63. 63.
    Green J, Ogunjobi OM, Ramage R, Stewart ASJ, McCurdy S, Noble R (1988) Application of the NG-(2,2,5,7,8-pentamethylchroman-6-sulphonyl) derivative of FMOC-arginine to peptide synthesis. Tetrahedron Lett 29:4341–4344CrossRefGoogle Scholar
  64. 64.
    Ramage R, Green J (1987) NG-2,2,5,7,8-pentamethylchroman-6-sulphonyl-L-arginine: a new acid labile derivative for peptide synthesis. Tetrahedron Lett 28:2287–2290CrossRefGoogle Scholar
  65. 65.
    Head E, Garzon-Rodriguez W, Johnson JK, Lott IT, Cotman CW, Glabe C (2001) Oxidation of Aβ and plaque biogenesis in Alzheimer’s disease and Down syndrome. Neurobiol Dis 8:792–806CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Martin Pattky
    • 1
  • Simone Nicolardi
    • 2
  • Beatrix Santiago-Schübel
    • 1
  • Daniel Sydes
    • 1
  • Yuri E. M. van der Burgt
    • 2
  • Antonia N. Klein
    • 3
  • Nan Jiang
    • 3
  • Jeannine Mohrlüder
    • 3
  • Karen Hänel
    • 3
  • Janine Kutzsche
    • 3
  • S. A. Funke
    • 3
    • 4
  • D. Willbold
    • 3
    • 5
  • S. Willbold
    • 1
  • C. Huhn
    • 1
    • 6
    Email author
  1. 1.Central Institute for Engineering, Electronics, and Analytics (ZEA): Analytics (ZEA-3), Forschungszentrum Jülich GmbHJülichGermany
  2. 2.Leiden University Medical Center, Center for Proteomics and MetabolomicsLeidenThe Netherlands
  3. 3.Institute of Complex Systems, Structural Biochemistry (ICS-6)Research Centre JülichJülichGermany
  4. 4.BioanalytikHochschule für Angewandte WissenschaftenCoburgGermany
  5. 5.Institut für Physikalische BiologieHeinrich-Heine-Universität DüsseldorfDüsseldorfGermany
  6. 6.Institute for Physical and Theoretical ChemistryEberhard Karls Universität TübingenTübingenGermany

Personalised recommendations