Peptidomics pp 107-118 | Cite as

Non-targeted Identification of d-Amino Acid-Containing Peptides Through Enzymatic Screening, Chiral Amino Acid Analysis, and LC-MS

  • Hua-Chia Tai
  • James W. Checco
  • Jonathan V. SweedlerEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1719)


d-Amino acid-containing peptides (DAACPs) in animals are a class of bioactive molecules formed via the posttranslational modification of peptides consisting of all-l-amino acid residues. Amino acid residue isomerization greatly impacts the function of the resulting DAACP. However, because isomerization does not change the peptide’s mass, this modification is difficult to detect by most mass spectrometry-based peptidomic approaches. Here we describe a method for the identification of DAACPs that can be used to systematically survey peptides extracted from a tissue sample in a non-targeted manner.

Key words

d-Amino acid-containing peptides Posttranslational modifications Bioactive peptides Peptide isomerization Chirality 



This work was supported by the National Institutes of Health, Award No. P30 DA018310 from the National Institute on Drug Abuse (NIDA) and Award No. 2 R01NS031609 from the National Institute of Neurological Disorders and Stroke (NINDS). J.W.C. was supported in part by a Beckman Institute Postdoctoral Fellowship, funded by a Beckman Foundation gift to the Beckman Institute for Advanced Science and Technology at the University of Illinois at Urbana-Champaign. The content is solely the responsibility of the authors and does not necessarily represent the official views of the funding agencies.


  1. 1.
    Bai L, Sheeley S, Sweedler JV (2009) Analysis of endogenous d-amino acid-containing peptides in Metazoa. Bioanal Rev 1(1):7–24. CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Ollivaux C, Soyez D, Toullec JY (2014) Biogenesis of d-amino acid containing peptides/proteins: where, when and how? J Pept Sci 20(8):595–612. CrossRefPubMedGoogle Scholar
  3. 3.
    Richter K, Egger R, Kreil G (1987) d-alanine in the frog skin peptide dermorphin is derived from l-alanine in the precursor. Science 238(4824):200–202CrossRefPubMedGoogle Scholar
  4. 4.
    Kamatani Y, Minakata H, Kenny PT, Iwashita T, Watanabe K, Funase K, Sun XP, Yongsiri A, Kim KH, Novales-Li P et al (1989) Achatin-I, an endogenous neuroexcitatory tetrapeptide from Achatina fulica Ferussac containing a d-amino acid residue. Biochem Biophys Res Commun 160(3):1015–1020CrossRefPubMedGoogle Scholar
  5. 5.
    Ohta N, Kubota I, Takao T, Shimonishi Y, Yasuda-Kamatani Y, Minakata H, Nomoto K, Muneoka Y, Kobayashi M (1991) Fulicin, a novel neuropeptide containing a d-amino acid residue isolated from the ganglia of Achatina fulica. Biochem Biophys Res Commun 178(2):486–493. CrossRefPubMedGoogle Scholar
  6. 6.
    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. CrossRefPubMedGoogle Scholar
  7. 7.
    Ollivaux C, Gallois D, Amiche M, Boscameric M, Soyez D (2009) Molecular and cellular specificity of post-translational aminoacyl isomerization in the crustacean hyperglycaemic hormone family. FEBS J 276(17):4790–4802. CrossRefPubMedGoogle Scholar
  8. 8.
    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. CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    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. CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    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. CrossRefPubMedGoogle Scholar
  11. 11.
    Koehbach J, Gruber CW, Becker C, Kreil DP, Jilek A (2016) MALDI TOF/TOF-based approach for the identification of d-amino acids in biologically active peptides and proteins. J Proteome Res 15(5):1487–1496. CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Sheeley SA, Miao H, Ewing MA, Rubakhin SS, Sweedler JV (2005) Measuring d-amino acid-containing neuropeptides with capillary electrophoresis. Analyst 130(8):1198–1203. CrossRefPubMedGoogle Scholar
  13. 13.
    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. CrossRefPubMedGoogle Scholar
  14. 14.
    Tao Y, Quebbemann NR, Julian RR (2012) Discriminating d-amino acid-containing peptide epimers by radical-directed dissociation mass spectrometry. Anal Chem 84(15):6814–6820. CrossRefPubMedGoogle Scholar
  15. 15.
    Livnat I, Tai HC, Jansson ET, Bai L, Romanova EV, Chen TT, Yu K, Chen SA, Zhang Y, Wang ZY, Liu DD, Weiss KR, Jing J, Sweedler JV (2016) A d-amino acid-containing neuropeptide discovery funnel. Anal Chem 88(23):11868–11876. CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Ewing MA, Wang J, Sheeley SA, Sweedler JV (2008) Detecting d-amino acid-containing neuropeptides using selective enzymatic digestion. Anal Chem 80(8):2874–2880. CrossRefPubMedGoogle Scholar
  17. 17.
    Bhushan R, Bruckner H (2004) Marfey’s reagent for chiral amino acid analysis: a review. Amino Acids 27(3–4):231–247. CrossRefPubMedGoogle Scholar
  18. 18.
    Yang CY, Yu K, Wang Y, Chen SA, Liu DD, Wang ZY, YN Su, Yang SZ, Chen TT, Livnat I, Vilim FS, Cropper EC, Weiss KR, Sweedler JV, Jing J (2016) Aplysia locomotion: network and behavioral actions of GdFFD, a d-amino acid-containing neuropeptide. PLoS One 11(1):e0147335. CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Finoulst I, Pinkse M, Van Dongen W, Verhaert P (2011) Sample preparation techniques for the untargeted LC-MS-based discovery of peptides in complex biological matrices. J Biomed Biotechnol 2011:245291. CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Conlon JM (2007) Purification of naturally occurring peptides by reversed-phase HPLC. Nat Protoc 2(1):191–197. CrossRefPubMedGoogle Scholar
  21. 21.
    Liardon R, Ledermann S, Ott U (1981) Determination of d-amino acids by deuterium labelling and selected ion monitoring. J Chromatogr A 203:385–395. CrossRefGoogle Scholar
  22. 22.
    Fountoulakis M, Lahm HW (1998) Hydrolysis and amino acid composition of proteins. J Chromatogr A 826(2):109–134CrossRefPubMedGoogle Scholar
  23. 23.
    Turner AJ (2013) Chapter 79—Aminopeptidase N In: Rawling ND, Salvesen G (eds) Handbook of Proteolytic Enzymes. Academic Press, London, pp 397–403.
  24. 24.
    Gill SC, von Hippel PH (1989) Calculation of protein extinction coefficients from amino acid sequence data. Anal Biochem 182(2):319–326.
  25. 25.
    Conibear AC, Daly NL, Craik DJ (2012) Quantification of small cyclic disulfide-rich peptides. Biopolymers 98(6):518–524. CrossRefPubMedGoogle Scholar
  26. 26.
    Anthis NJ, Clore GM (2013) Sequence-specific determination of protein and peptide concentrations by absorbance at 205 nm. Protein Sci 22(6):851–858. CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Murai Y, Wang L, Masuda K, Sakihama Y, Hashidoko Y, Hatanaka Y, Hashimoto M (2013) Rapid and controllable hydrogen/deuterium exchange on aromatic rings of α-amino acids and peptides. Eur J Org Chem 2013(23):5111–5116. CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2018

Authors and Affiliations

  • Hua-Chia Tai
    • 1
  • James W. Checco
    • 1
  • Jonathan V. Sweedler
    • 1
    Email author
  1. 1.Department of ChemistryBeckman Institute for Advanced Science and Technology, University of Illinois at Urbana—ChampaignUrbanaUSA

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