Skip to main content

Advertisement

SpringerLink
Log in

Separation of peptide fragments of a protein kinase C substrate fused to a β-hairpin by capillary electrophoresis

  • Research Paper
  • Published:
Analytical and Bioanalytical Chemistry Aims and scope Submit manuscript

Abstract

Synthetic peptides incorporating well-folded β-hairpin peptides possess advantages in a variety of cell biology applications by virtue of increased resistance to proteolytic degradation. In this study, the WKpG β-hairpin peptide fused to a protein kinase C (PKC) substrate was synthesized, and capillary-electrophoretic separation conditions for this peptide and its proteolytic fragments were developed. Fragments of WKpG-PKC were generated by enzymatic treatment with trypsin and Pronase E to produce standards for identification of degradation fragments in a cellular lysate. A simple buffer system of 250 mM H3PO4, pH 1.5 enabled separation of WKpG-PKC and its fragments by capillary electrophoresis in less than 16 min. Using a cellular lysate produced from Ba/F3 cells, the β-hairpin-conjugated substrate and its PKCα-phosphorylated product could be detected and separated from peptidase-generated fragments produced in a cell lysate. The method has potential application for identification and quantification of WKpG-PKC and its fragments in complex biological systems when the peptide is used as a reporter to assay PKC activity.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

Abbreviations

6-FAM:

6-Carboxifluorescein

Ac:

Acetyl

CE-LIF:

Capillary electrophoresis with laser-induced fluorescence

DBU:

1,8-Diazobicyclo[5.4.0]undec-7-ene

DIPEA:

N,N ′-Diisopropylethylamine

DMF:

N,N-Dimethylformamide

HBTU:

O-(Benzotriazol-1-yl)-N,N,N ′,N ′-tetramethyluronium hexafluorophosphate

HOBt:

N-Hydroxybenzotriazole

MALDI-MS:

Matrix-assisted laser desorption ionization mass spectrometry time-of-flight

MM:

Multiple myeloma

NMP:

N-Methylmorpholine

PBS:

Phosphate buffered saline

PEG:

Polyethylene glycol

PKC:

Protein kinase C

PyBOP:

Bromo-tris-pyrrolidono phosphonium hexafluorophosphate

PyClock:

6-Chloro-benzotriazole-1-yloxy-tris-pyrrolidinophosphonium hexafluorophosphate

TFA:

Trifluoroacetic acid

TIPS:

Triisopropylsilane

References

  1. Gellman SH (1998) Minimal model systems for beta sheet secondary structure in proteins. Curr Opin Chem Biol 2(6):717–725

    Article  CAS  Google Scholar 

  2. Syud FA, Stanger HE, Gellman SH (2001) Interstrand side chain-side chain interactions in a designed beta-hairpin: significance of both lateral and diagonal pairings. J Am Chem Soc 123(36):8667–8677

    Article  CAS  Google Scholar 

  3. Cochran AG, Skelton NJ, Starovasnik MA (2001) Tryptophan zippers: stable, monomeric beta-hairpins. Proc Natl Acad Sci U S A 98(10):5578–5583

    Article  CAS  Google Scholar 

  4. Ramirez-Alvarado M, Blanco FJ, Serrano L (1996) De novo design and structural analysis of a model beta-hairpin peptide system. Nat Struct Biol 3(7):604–612

    Article  CAS  Google Scholar 

  5. Hutchinson EG, Sessions RB, Thornton JM, Woolfson DN (1998) Determinants of strand register in antiparallel beta-sheets of proteins. Protein Sci 7(11):2287–2300

    Article  CAS  Google Scholar 

  6. Watts TH, Gariepy J, Schoolnik GK, Mcconnell HM (1985) T-cell activation by peptide antigen—effect of peptide sequence and method of antigen presentation. Proc Natl Acad Sci U S A 82(16):5480–5484

    Article  CAS  Google Scholar 

  7. Proctor A, Wang Q, Lawrence DS, Allbritton NL (2012) Development of a peptidase-resistant substrate for single-cell measurement of protein kinase B activation. Anal Chem 84(16):7195–7202

    Article  CAS  Google Scholar 

  8. Phillips RM, Bair E, Lawrence DS, Sims CE, Allbritton NL (2013) Measurement of protein tyrosine phosphatase activity in single cells by capillary electrophoresis. Anal Chem 85(12):6136–6142

    Article  CAS  Google Scholar 

  9. Proctor A, Wang Q, Lawrence DS, Allbritton NL (2012) Metabolism of peptide reporters in cell lysates and single cells. Analyst 137(13):3028–3038

    Article  CAS  Google Scholar 

  10. Yang S, Proctor A, Cline LL, Houston KM, Waters ML, Allbritton NL (2013) Beta-turn sequences promote stability of peptide substrates for kinases within the cytosolic environment. Analyst 138(15):4305–4311

    Article  CAS  Google Scholar 

  11. Pasut G, Veronese FM (2009) PEG conjugates in clinical development or use as anticancer agents: an overview. Adv Drug Deliv Rev 61(13):1177–1188

    Article  CAS  Google Scholar 

  12. Frackenpohl J, Arvidsson PI, Schreiber JV, Seebach D (2001) The outstanding biological stability of beta- and gamma-peptides toward proteolytic enzymes: an in vitro investigation with fifteen peptidases. Chembiochem 2(6):445–455

    Article  CAS  Google Scholar 

  13. Hook DF, Bindschadler P, Mahajan YR, Sebesta R, Kast P, Seebach D (2005) The proteolytic stability of ‘designed’ beta-peptides containing alpha-peptide-bond mimics and of mixed alpha, beta-peptides: application to the construction of MHC-binding peptides. Chem Biodivers 2(5):591–632

    Article  CAS  Google Scholar 

  14. Cline LL, Waters ML (2009) The structure of well-folded beta-hairpin peptides promotes resistance to peptidase degradation. Biopolymers 92(6):502–507

    Article  CAS  Google Scholar 

  15. Porter EA, Weisblum B, Gellman SH (2002) Mimicry of host-defense peptides by unnatural oligomers: antimicrobial beta-peptides. J Am Chem Soc 124(25):7324–7330

    Article  CAS  Google Scholar 

  16. Waters ML (2004) Aromatic interactions in peptides: impact on structure and function. Biopolymers 76(5):435–445

    Article  CAS  Google Scholar 

  17. Cheng Z, Campbell RE (2006) Assessing the structural stability of designed beta-hairpin peptides in the cytoplasm of live cells. Chembiochem 7(8):1147–1150

    Article  CAS  Google Scholar 

  18. Cheng Z, Miskolzie M, Campbell RE (2007) In vivo screening identifies a highly folded beta-hairpin peptide with a structured extension. ChemBioChem 8(8):880–883

    Article  CAS  Google Scholar 

  19. Saikumari YK, Ravindra G, Balaram P (2006) Structure formation in short designed peptides probed by proteolytic cleavage. Protein Pept Lett 13(5):471–476

    Article  CAS  Google Scholar 

  20. Hughes RM, Waters ML (2005) Influence of N-methylation on a cation-pi interaction produces a remarkably stable beta-hairpin peptide. J Am Chem Soc 127(18):6518–6519

    Article  CAS  Google Scholar 

  21. Fesinmeyer RM, Hudson FM, Andersen NH (2004) Enhanced hairpin stability through loop design: the case of the protein G B1 domain hairpin. J Am Chem Soc 126(23):7238–7243

    Article  CAS  Google Scholar 

  22. Riemen AJ, Waters ML (2009) Design of highly stabilized beta-hairpin peptides through cation-pi interactions of lysine and N-methyllysine with an aromatic pockets. Biochemistry 48(7):1525–1531

    Article  CAS  Google Scholar 

  23. Cooper WJ, Waters ML (2005) Turn residues in beta-hairpin peptides as points for covalent modification. Org Lett 7(18):3825–3828

    Article  CAS  Google Scholar 

  24. Hughes RM, Benshoff ML, Waters ML (2007) Effects of chain length and N-methylation on a cation-pi interaction in a beta-hairpin peptide. Chem Eur J 13(20):5753–5764

    Article  CAS  Google Scholar 

  25. Zigoneanu IG, Pielak GJ (2012) Interaction of alpha-synuclein and a cell penetrating fusion peptide with higher eukaryotic cell membranes assessed by 19F NMR. Mol Pharm 9(4):1024–1029

    Article  CAS  Google Scholar 

  26. Yang S (2012) Development of peptidase resistant reporters for intracellular enzymatic activity. Dissertation, University of North Carolina-Chapel Hill, pp. 221

  27. Wang K, Jiang D, Sims CE, Allbritton NL (2012) Separation of fluorescently labeled phosphoinositides and sphingolipids by capillary electrophoresis. J Chromatogr B Anal Technol Biomed Life Sci 907:79–86

    Article  CAS  Google Scholar 

  28. Gilar M, Belenky A, Smisek DL, Bourque A, Cohen AS (1997) Kinetics of phosphorothioate oligonucleotide metabolism in biological fluids. Nucleic Acids Res 25(18):3615–3620

    Article  CAS  Google Scholar 

  29. Burgi D, Smith AJ (2001) Capillary electrophoresis of proteins and peptides. Curr Protoc Mol Biol 10:10.20.1–10.20.13

  30. Righetti PG, Sebastiano R, Citterio A (2013) Capillary electrophoresis and isoelectric focusing in peptide and protein analysis. Proteomics 13:325–340

    Article  CAS  Google Scholar 

  31. Kasicka V (2014) Recent developments in capillary and microchip electroseparations of peptides (2011–2013). Electrophoresis 35:69–95

    Article  CAS  Google Scholar 

  32. Dickinson AJ, Armistead PM, Allbritton NL (2013) Automated capillary electrophoresis system for fast single-cell analysis. Anal Chem 85(9):4797–4804

    Article  CAS  Google Scholar 

  33. Li YS, Zhong XX, Rider AE, Furman SA, Ostrikov K (2014) Fast, energy-efficient synthesis of luminescent carbon quantum dots. Green Chem 16(5):2566–2570

    Article  CAS  Google Scholar 

  34. Dolan JW (2002) Peak tailing and resolution. LCGC Eur 15(6):334–337

    CAS  Google Scholar 

  35. Lee KJ, Mwongela SM, Kottegoda S, Borland L, Nelson AR, Sims CE, Allbritton NL (2008) Determination of sphingosine kinase activity for cellular signaling studies. Anal Chem 80(5):1620–1627

    Article  CAS  Google Scholar 

  36. Funk GM, Hunt CE, Epps DE, Brown PK (1986) Use of a rapid and highly sensitive fluorescamine-based procedure for the assay of plasma lipoproteins. J Lipid Res 27(7):792–795

    CAS  Google Scholar 

  37. Nishikawa K, Toker A, Johannes FJ, Songyang Z, Cantley LC (1997) Determination of the specific substrate sequence motifs of protein kinase C isozymes. J Biol Chem 272(2):952–960

    Article  CAS  Google Scholar 

  38. Yaffe MB, Leparc GG, Lai J, Obata T, Volinia S, Cantley LC (2001) A motif-based profile scanning approach for genome-wide prediction of signaling pathways. Nat Biotechnol 19(4):348–353

    Article  CAS  Google Scholar 

  39. Cline LL, Waters ML (2009) Design of a beta-hairpin peptide-intercalator conjugate for simultaneous recognition of single stranded and double stranded regions of RNA. Org Biomol Chem 7(22):4622–4630

    Article  CAS  Google Scholar 

  40. Whitcomb DC, Lowe ME (2007) Human pancreatic digestive enzymes. Dig Dis Sci 52(1):1–17

    Article  CAS  Google Scholar 

  41. Zenobi R, Knochenmuss R (1998) Ion formation in MALDI mass spectrometry. Mass Spectrom Rev 17(5):337–366

    Article  CAS  Google Scholar 

  42. Cifuentes A, Poppe H (1995) Effect of pH and ionic strength of running buffer on peptide behavior in capillary electrophoresis: theoretical calculation and experimental evaluation. Electrophoresis 16:516–524

    Article  CAS  Google Scholar 

  43. Koval D, Kasicka V, Jiracek J, Collinsova M (2003) Physicochemical characterization of phosphinic pseudopeptides by capillary zone electrophoresis in highly acidic background electrolytes. Electrophoresis 24:774–781

    Article  CAS  Google Scholar 

  44. Mccormick RM (1988) Capillary zone electrophoretic separation of peptides and proteins using low pH buffers in modified silica capillaries. Anal Chem 60:2322–2328

    Article  CAS  Google Scholar 

  45. Righetti PG, Bossi A, Olivieri E, Gelfi C (1999) Capillary electrophoresis of peptides and proteins in acidic, isoelectric buffers: recent developments. J Biochem Biophys Methods 40(1–2):1–15

    Article  CAS  Google Scholar 

Download references

Acknowledgments

The authors thank Effrat Fayer and Marcey Waters for helpful discussions. Funding for this research was provided by the National Institutes of Health (CA177993 and EY024556 to NLA and CA186748 to IGZ).

Conflict of interest

The authors report no conflicts of interest in this work.

Author information

Authors and Affiliations

  1. Department of Biomedical Engineering, University of North Carolina, Chapel Hill, NC, 27599, USA

    Imola G. Zigoneanu & Nancy L. Allbritton

  2. North Carolina State University, Raleigh, NC, 27695, USA

    Imola G. Zigoneanu & Nancy L. Allbritton

  3. Department of Chemistry, University of North Carolina, Chapel Hill, NC, 27599, USA

    Christopher E. Sims & Nancy L. Allbritton

Authors
  1. Imola G. Zigoneanu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Christopher E. Sims
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Nancy L. Allbritton
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Nancy L. Allbritton.

Electronic supplementary material

Below is the link to the electronic supplementary material.

ESM 1

(PDF 440 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Zigoneanu, I.G., Sims, C.E. & Allbritton, N.L. Separation of peptide fragments of a protein kinase C substrate fused to a β-hairpin by capillary electrophoresis. Anal Bioanal Chem 407, 8999–9008 (2015). https://doi.org/10.1007/s00216-015-9065-8

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00216-015-9065-8

Keywords

Search

Navigation