Skip to main content
SpringerLink
Log in

Site-specific Localization of D-Amino Acids in Bioactive Peptides by Ion Mobility Spectrometry

  • Protocol
  • First Online:
Analysis of Post-Translational Modifications and Proteolysis in Neuroscience

Part of the book series: Neuromethods ((NM,volume 114))

Abstract

In this study, we describe a site-specific strategy to rapidly and precisely localize D-amino acids in peptides by ion mobility spectrometry (IMS) analysis of mass spectrometry (MS)-generated epimeric fragment ions. Briefly, the D/L-peptide epimers are separated by online reversed-phase liquid chromatography (LC) and fragmented by collision induced dissociation (CID), followed by IMS analysis. The epimeric fragment ions resulting from D/L-peptide epimers exhibit arrival time differences, thus showing different mobility in IMS. The arrival time shift between the epimeric fragment ions is used as criteria to localize the D-amino acid substitution. We provide the technical details on sample preparation, LC-tandem mass spectrometry analysis, data processing, and collisional cross-section calibration.

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

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 109.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Iida T, Santa T, Toriba A, Imai K (2001) Amino acid sequence and D/L-configuration determination methods for D-amino acid-containing peptides in living organisms. Biomed Chromatogr 15(5):319–327

    Article  CAS  PubMed  Google Scholar 

  2. Bai L, Romanova EV, Sweedler JV (2011) Distinguishing endogenous D-amino acid-containing neuropeptides in individual neurons using tandem mass spectrometry. Anal Chem 83(7):2794–2800

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Bai L, Sheeley S, Sweedler JV (2009) Analysis of endogenous D-amino acid-containing peptides in metazoa. Bioanal Rev 1(1):7–24

    Article  PubMed  PubMed Central  Google Scholar 

  4. Bai L, Livnat I, Romanova EV, Alexeeva V, Yau PM, Vilim FS, Weiss KR, Jing J, Sweedler JV (2013) Characterization of GdFFD, a D-amino acid-containing neuropeptide that functions as an extrinsic modulator of the Aplysia feeding circuit. J Biol Chem 288(46):32837–32851

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Adams CM, Zubarev RA (2005) Distinguishing and quantifying peptides and proteins containing D-amino acids by tandem mass spectrometry. Anal Chem 77(14):4571–4580

    Article  CAS  PubMed  Google Scholar 

  6. Hurtado PP, O’Connor PB (2012) Differentiation of isomeric amino acid residues in proteins and peptides using mass spectrometry. Mass Spectrom Rev 31(6):609–625

    Article  CAS  PubMed  Google Scholar 

  7. Tao WA, Cooks RG (2001) Parallel reactions for enantiomeric quantification of peptides by mass spectrometry this work was supported by the U.S. Department of energy, office of energy research. W.A.T. Acknowledges fellowship support from triangle pharmaceuticals. Angew Chem Int Ed Engl 40(4):757–760

    Article  CAS  PubMed  Google Scholar 

  8. Serafin SV, Maranan R, Zhang K, Morton TH (2005) Mass spectrometric differentiation of linear peptides composed of L-amino acids from isomers containing one D-amino acid residue. Anal Chem 77(17):5480–5487

    Article  CAS  PubMed  Google Scholar 

  9. Sachon E, Clodic G, Galanth C, Amiche M, Ollivaux C, Soyez D, Bolbach G (2009) D-amino acid detection in peptides by MALDI-TOF-TOF. Anal Chem 81(11):4389–4396

    Article  CAS  PubMed  Google Scholar 

  10. Soyez D, Van Herp F, Rossier J, Le Caer JP, Tensen CP, Lafont R (1994) Evidence for a conformational polymorphism of invertebrate neurohormones. D-amino acid residue in crustacean hyperglycemic peptides. J Biol Chem 269(28):18295–18298

    CAS  PubMed  Google Scholar 

  11. Soyez D, Toullec JY, Montagne N, Ollivaux C (2011) Experimental strategies for the analysis of D-amino acid containing peptides in crustaceans: a review. J Chromatogr B Analyt Technol Biomed Life Sci 879(29):3102–3107

    Article  CAS  PubMed  Google Scholar 

  12. Buczek O, Yoshikami D, Bulaj G, Jimenez EC, Olivera BM (2005) Post-translational amino acid isomerization: a functionally important D-amino acid in an excitatory peptide. J Biol Chem 280(6):4247–4253

    Article  CAS  PubMed  Google Scholar 

  13. Serrano L, Grousset E, Charmantier G, Spanings-Pierrot C (2004) Occurrence of L- and D-crustacean hyperglycemic hormone isoforms in the eyestalk X-organ/sinus gland complex during the ontogeny of the crayfish Astacus leptodactylus. J Histochem Cytochem 52(9):1129–1140

    Article  CAS  PubMed  Google Scholar 

  14. Iida T, Matsunaga H, Santa T, Fukushima T, Homma H, Imai K (1998) Amino acid sequence and D/L-configuration determination of peptides utilizing liberated N-terminus phenylthiohydantoin amino acids. J Chromatogr A 813(2):267–275

    Article  CAS  PubMed  Google Scholar 

  15. Bohrer BC, Merenbloom SI, Koeniger SL, Hilderbrand AE, Clemmer DE (2008) Biomolecule analysis by ion mobility spectrometry. Annu Rev Anal Chem (Palo Alto Calif) 1:293–327

    Article  CAS  Google Scholar 

  16. Enders JR, McLean JA (2009) Chiral and structural analysis of biomolecules using mass spectrometry and ion mobility-mass spectrometry. Chirality 21(Suppl 1):E253–E264

    Article  CAS  PubMed  Google Scholar 

  17. Verbeck GF, Ruotolo BT, Sawyer HA, Gillig KJ, Russell DH (2002) A fundamental introduction to ion mobility mass spectrometry applied to the analysis of biomolecules. J Biomol Tech 13(2):56–61

    PubMed  PubMed Central  Google Scholar 

  18. Shvartsburg AA, Tang K, Smith RD (2009) Two-dimensional ion mobility analyses of proteins and peptides. Methods Mol Biol 492:417–445

    Article  CAS  PubMed  Google Scholar 

  19. Fenn LS, McLean JA (2008) Biomolecular structural separations by ion mobility-mass spectrometry. Anal Bioanal Chem 391(3):905–909

    Article  CAS  PubMed  Google Scholar 

  20. Lanucara F, Holman SW, Gray CJ, Eyers CE (2014) The power of ion mobility-mass spectrometry for structural characterization and the study of conformational dynamics. Nat Chem 6(4):281–294

    Article  CAS  PubMed  Google Scholar 

  21. Jurneczko E, Barran PE (2011) How useful is ion mobility mass spectrometry for structural biology? The relationship between protein crystal structures and their collision cross sections in the gas phase. Analyst 136(1):20–28

    Article  CAS  PubMed  Google Scholar 

  22. Jia C, Hui L, Cao W, Lietz CB, Jiang X, Chen R, Catherman AD, Thomas PM, Ge Y, Kelleher NL, Li L (2012) High-definition de novo sequencing of crustacean hyperglycemic hormone (CHH)-family neuropeptides. Mol Cell Proteomics 11(12):1951–1964

    Article  PubMed  PubMed Central  Google Scholar 

  23. Hui L, Cunningham R, Zhang Z, Cao W, Jia C, Li L (2011) Discovery and characterization of the Crustacean hyperglycemic hormone precursor related peptides (CPRP) and orcokinin neuropeptides in the sinus glands of the blue crab Callinectes sapidus using multiple tandem mass spectrometry techniques. J Proteome Res 10(9):4219–4229

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Jia C, Lietz CB, Yu Q, Li L (2014) Site-specific characterization of D-amino acid containing peptide epimers by ion mobility spectrometry. Anal Chem 86(6):2972–2981

    Article  CAS  PubMed  Google Scholar 

  25. Bush MF, Campuzano ID, Robinson CV (2012) Ion mobility mass spectrometry of peptide ions: effects of drift gas and calibration strategies. Anal Chem 84(16):7124–7130

    Article  CAS  PubMed  Google Scholar 

  26. Ruotolo BT, Benesch JL, Sandercock AM, Hyung SJ, Robinson CV (2008) Ion mobility-mass spectrometry analysis of large protein complexes. Nat Protoc 3(7):1139–1152

    Article  CAS  PubMed  Google Scholar 

  27. Lietz CB, Yu Q, Li L (2014) Large-scale collision cross-section profiling on a traveling wave Ion mobility mass spectrometer. J Am Soc Mass Spectrom 25(12):2009–2019

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

This work is supported in part by the National Institutes of Health (NIH) grant (R01DK071801 to LL) and the National Science Foundation grant (CHE-1413596 to LL). LL acknowledges an H. I. Romnes Faculty Research Fellowship from UW-Madison, Tianjin 1000 Talent Plan from Tianjin China and Changjiang Professorship from the Chinese Ministry of Education. C.L. acknowledges an NIH-supported Chemistry Biology Interface Training Program Predoctoral Fellowship (grant number T32-GM008505) and an NSF Graduate Research Fellowship (DGE-1256259). We are grateful to Prof. Jonathan V. Sweedler, Dr. Lu Bai, and Itamar Livnat (UIUC) for helpful discussions and insightful suggestions on this project.

Author information

Authors and Affiliations

  1. School of Pharmacy, University of Wisconsin–Madison, 777 Highland Avenue, Madison, WI, 53705-2222, USA

    Chenxi Jia, Qing Yu & Lingjun Li

  2. Department of Chemistry, University of Wisconsin–Madison, Madison, WI, USA

    Christopher B. Lietz & Lingjun Li

  3. School of Life Sciences, Tianjin University, Tianjin, China

    Lingjun Li

  4. National Center for Protein Sciences-Beijing, and Beijing Proteome Research Center, 38 Science Park Road, Changping District, Beijing, 102206, China

    Chenxi Jia

Authors
  1. Chenxi Jia
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Christopher B. Lietz
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Qing Yu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Lingjun Li
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Lingjun Li .

Editor information

Editors and Affiliations

  1. Department of Biology, University of Wisconsin-Stout, Menomonie, Wisconsin, USA

    Jennifer Elizabeth Grant

  2. Department of Microbiology, Biochemistry and Molecular Genetics, Rutgers University—New Jersey Medical School Cancer Center Newark, Newark, New Jersey, USA

    Hong Li

Rights and permissions

Reprints and permissions

Copyright information

© 2016 Springer Science+Business Media New York

About this protocol

Cite this protocol

Jia, C., Lietz, C.B., Yu, Q., Li, L. (2016). Site-specific Localization of D-Amino Acids in Bioactive Peptides by Ion Mobility Spectrometry. In: Grant, J., Li, H. (eds) Analysis of Post-Translational Modifications and Proteolysis in Neuroscience. Neuromethods, vol 114. Humana Press, New York, NY. https://doi.org/10.1007/7657_2015_82

Download citation

  • DOI: https://doi.org/10.1007/7657_2015_82

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-3470-6

  • Online ISBN: 978-1-4939-3472-0

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation