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

  • Chenxi Jia
  • Christopher B. Lietz
  • Qing Yu
  • Lingjun LiEmail author
Part of the Neuromethods book series (NM, volume 114)


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.


Neuropeptide Peptidomics De novo sequencing Mass spectrometry Crustacean Ion mobility 



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.


  1. 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):319–327CrossRefPubMedGoogle Scholar
  2. 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):2794–2800CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Bai L, Sheeley S, Sweedler JV (2009) Analysis of endogenous D-amino acid-containing peptides in metazoa. Bioanal Rev 1(1):7–24CrossRefPubMedPubMedCentralGoogle Scholar
  4. 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):32837–32851CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Adams CM, Zubarev RA (2005) Distinguishing and quantifying peptides and proteins containing D-amino acids by tandem mass spectrometry. Anal Chem 77(14):4571–4580CrossRefPubMedGoogle Scholar
  6. 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):609–625CrossRefPubMedGoogle Scholar
  7. 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):757–760CrossRefPubMedGoogle Scholar
  8. 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):5480–5487CrossRefPubMedGoogle Scholar
  9. 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):4389–4396CrossRefPubMedGoogle Scholar
  10. 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):18295–18298PubMedGoogle Scholar
  11. 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):3102–3107CrossRefPubMedGoogle Scholar
  12. 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):4247–4253CrossRefPubMedGoogle Scholar
  13. 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):1129–1140CrossRefPubMedGoogle Scholar
  14. 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):267–275CrossRefPubMedGoogle Scholar
  15. 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) 1:293–327CrossRefGoogle Scholar
  16. 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–E264CrossRefPubMedGoogle Scholar
  17. 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):56–61PubMedPubMedCentralGoogle Scholar
  18. 18.
    Shvartsburg AA, Tang K, Smith RD (2009) Two-dimensional ion mobility analyses of proteins and peptides. Methods Mol Biol 492:417–445CrossRefPubMedGoogle Scholar
  19. 19.
    Fenn LS, McLean JA (2008) Biomolecular structural separations by ion mobility-mass spectrometry. Anal Bioanal Chem 391(3):905–909CrossRefPubMedGoogle Scholar
  20. 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):281–294CrossRefPubMedGoogle Scholar
  21. 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):20–28CrossRefPubMedGoogle Scholar
  22. 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):1951–1964CrossRefPubMedPubMedCentralGoogle Scholar
  23. 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):4219–4229CrossRefPubMedPubMedCentralGoogle Scholar
  24. 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):2972–2981CrossRefPubMedGoogle Scholar
  25. 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):7124–7130CrossRefPubMedGoogle Scholar
  26. 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):1139–1152CrossRefPubMedGoogle Scholar
  27. 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):2009–2019CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Chenxi Jia
    • 1
    • 4
  • Christopher B. Lietz
    • 2
  • Qing Yu
    • 1
  • Lingjun Li
    • 1
    • 2
    • 3
    Email author
  1. 1.School of PharmacyUniversity of Wisconsin–MadisonMadisonUSA
  2. 2.Department of ChemistryUniversity of Wisconsin–MadisonMadisonUSA
  3. 3.School of Life SciencesTianjin UniversityTianjinChina
  4. 4.National Center for Protein Sciences-Beijing, and Beijing Proteome Research CenterBeijingChina

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