Skip to main content
SpringerLink
Log in

Investigation of bn-44 Peptide Fragments Using High Resolution Mass Spectrometry and Isotope Labeling

  • Research Article
  • Published:
Journal of The American Society for Mass Spectrometry

Abstract

An N-terminal deuterohemin-containing hexapeptide (DhHP-6) was designed as a short peptide cytochrome c (Cyt c) mimetic to study the effect of N-terminal charge on peptide fragmentation pathways. This peptide gave different dissociation patterns than normal tryptic peptides. Upon collision-induced dissociation (CID) with an ion trap mass spectrometer, the singly charged peptide ion containing no added proton generated abundant and characteristic bn-44 ions instead of bn-28 (an) ions. Studies by high resolution mass spectrometry (HRMS) and isotope labeling indicate that elimination of 44 Da fragments from b ions occurs via two different pathways: (1) loss of CH3CHO (44.0262) from a Thr side chain; (2) loss of CO2 (43.9898) from the oxazolone structure in the C-terminus. A series of analogues were designed and analyzed. The experimental results combined with Density Functional Theory (DFT) calculations on the proton affinity of the deuteroporphyrin demonstrate that the production of these novel bn-44 ions is related to the N-terminal charge via a charge-remote rather than radical-directed fragmentation pathway.

ᅟ

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

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Scheme 1

Similar content being viewed by others

References

  1. Aebersold, R., Goodlett, D.R.: Mass spectrometry in proteomics. Chem. Rev. 101, 269–296 (2001)

    Article  CAS  Google Scholar 

  2. Seidler, J., Zinn, N., Boehm, M.E., Lehmann, W.D.: De novo sequencing of peptides by MS/MS. Proteomics 10, 634–649 (2010)

    Article  CAS  Google Scholar 

  3. Medzihradszky, K.F., Chalkley, R.J.: Lessons in de novo peptide sequencing by tandem mass spectrometry. Mass Spectrom. Rev. 1–21 (2013). doi:10.1002/mas.21406

  4. Escobar, H., Reyes-Vargas, E., Jensen, P.E., Delgado, J.C., Crockett, D.K.: Utility of characteristic QTOF MS/MS fragmentation for MHC class I peptides. J. Proteome Res. 10, 2494–2507 (2011)

    Article  CAS  Google Scholar 

  5. Mouls, L., Aubagnac, J.L., Martinez, J., Enjalbal, C.: Low energy peptide fragmentations in an ESI-Q-Tof type mass spectrometer. J. Proteome Res. 6, 1378–1391 (2007)

    Article  CAS  Google Scholar 

  6. Dongre, A.R., Jones, J.L., Somogyi, Á., Wysocki, V.H.: Influence of peptide composition, gas-phase basicity, and chemical modification on fragmentation efficiency: evidence for the mobile proton model. J. Am. Chem. Soc. 118, 8365–8374 (1996)

    Article  CAS  Google Scholar 

  7. Paizs, B., Suhai, S.: Fragmentation pathways of protonated peptides. Mass Spectrom. Rev. 24, 508–548 (2005)

    Article  CAS  Google Scholar 

  8. Tabb, D.L., Huang, Y., Wysocki, V.H., Yates, J.R.: Influence of basic residue content on fragment ion peak intensities in low-energy collision-induced dissociation spectra of peptides. Anal. Chem. 76, 1243–1248 (2004)

    Article  CAS  Google Scholar 

  9. Huang, Y., Triscari, J.M., Tseng, G.C., Pasa-Tolic, L., Lipton, M.S., Smith, R.D., Wysocki, V.H.: Statistical characterization of the charge state and residue dependence of low-energy CID peptide dissociation patterns. Anal. Chem. 77, 5800–5813 (2005)

    Article  CAS  Google Scholar 

  10. Laskin, J., Yang, Z., Song, T., Lam, C., Chu, I.K.: Effect of the basic residue on the energetics, dynamics, and mechanisms of gas-phase fragmentation of protonated peptides. J. Am. Chem. Soc. 132, 16006–16016 (2010)

    Article  CAS  Google Scholar 

  11. Tsaprailis, G., Nair, H., Somogyi, Á., Wysocki, V.H., Zhong, W., Futrell, J.H., Summerfield, S.G., Gaskell, S.J.: Influence of secondary structure on the fragmentation of protonated peptides. J. Am. Chem. Soc. 121, 5142–5154 (1999)

    Article  CAS  Google Scholar 

  12. Schweppe, R.E., Haydon, C.E., Lewis, T.S., Resing, K.A., Ahn, N.G.: The characterization of protein post-translational modifications by mass spectrometry. Acc. Chem. Res. 36, 453–461 (2003)

    Article  CAS  Google Scholar 

  13. Sprung, R., Chen, Y., Zhang, K., Cheng, D., Zhang, T., Peng, J., Zhao, Y.: Identification and validation of eukaryotic aspartate and glutamate methylation in proteins. J. Proteome Res. 7, 1001–1006 (2008)

    Article  CAS  Google Scholar 

  14. Palumbo, A.M., Tepe, J.J., Reid, G.E.: Mechanistic insights into the multistage gas-phase fragmentation behavior of phosphoserine- and phosphothreonine-containing peptides. J. Proteome Res. 7, 771–779 (2008)

    Article  CAS  Google Scholar 

  15. Reid, G.E., Roberts, K.D., Kapp, E.A., Simpson, R.J.: Statistical and mechanistic approaches to understanding the gas-phase fragmentation behavior of methionine sulfoxide containing peptides. J. Proteome Res. 3, 751–759 (2004)

    Article  CAS  Google Scholar 

  16. Harrison, A.G.: To b or not to b: the ongoing saga of peptide b ions. Mass Spectrom. Rev. 28, 640–654 (2009)

    Article  CAS  Google Scholar 

  17. Polfer, N.C., Oomens, J., Suhai, S., Paizs, B.: Spectroscopic and theoretical evidence for oxazolone ring formation in collision-induced dissociation of peptides. J. Am. Chem. Soc. 127, 17154–17155 (2005)

    Article  CAS  Google Scholar 

  18. Gu, C., Tsaprailis, G., Breci, L., Wysocki, V.H.: Selective gas-phase cleavage at the peptide bond C-terminal to aspartic acid in fixed-charge derivatives of Asp-containing peptides. Anal. Chem. 72, 5804–5813 (2000)

    Article  CAS  Google Scholar 

  19. Wang, B., Shang, J.Z., Qin, Y.J., Yan, B.N., Guo, X.H.: Differentiation of α-or β-aspartic isomers in the heptapeptides by the fragments of [M+ Na]+ using ion trap tandem mass spectrometry. J. Am. Soc. Mass Spectrom. 22, 1453–1462 (2011)

    Article  Google Scholar 

  20. Switzar, L., Giera, M., Niessen, W.M.: Protein digestion: an overview of the available techniques and recent developments. J. Proteome Res. 12, 1067–1077 (2013)

    Article  CAS  Google Scholar 

  21. Bythell, B.J., Csonka, I.P., Suhai, S., Barofsky, D.F., Paizs, B.: Gas-phase structure and fragmentation pathways of singly protonated peptides with N-terminal arginine. J. Phys. Chem. B 114, 15092–15105 (2010)

    Article  CAS  Google Scholar 

  22. Bythell, B.J., Suhai, S., Somogyi, Á., Paizs, B.: Proton-driven amide bond-cleavage pathways of gas-phase peptide ions lacking mobile protons. J. Am. Chem. Soc. 131, 14057–14065 (2009)

    Article  CAS  Google Scholar 

  23. Guan, S., Li, P., Luo, J., Li, Y., Huang, L., Wang, G., Zhu, L., Fan, H., Li, W., Wang, L.: A deuterohemin peptide extends lifespan and increases stress resistance in Caenorhabditis elegans. Free Radic. Res. 44, 813–820 (2010)

    Article  CAS  Google Scholar 

  24. He, F., Hendrickson, C.L., Marshall, A.G.: Unequivocal determination of metal atom oxidation state in naked heme proteins: Fe (III) myoglobin, Fe (III) cytochrome c, Fe (III) cytochrome b5, and Fe (III) cytochrome b5 L47R. J. Am. Soc. Mass Spectrom. 11, 120–126 (2000)

    Article  CAS  Google Scholar 

  25. Yang, F., Bogdanov, B., Strittmatter, E.F., Vilkov, A.N., Gritsenko, M., Shi, L., Elias, D.A., Ni, S., Romine, M., Paša-Tolic, L.: Characterization of purified c-type heme-containing peptides and identification of c-type heme-attachment sites in Shewanella oneidenis cytochromes using mass spectrometry. J. Proteome Res. 4, 846–854 (2005)

    Article  CAS  Google Scholar 

  26. Pashynska, V.A., Van den Heuvel, H., Claeys, M., Kosevich, M.V.: Characterization of noncovalent complexes of antimalarial agents of the artemisinin-type and FE (III)-heme by electrospray mass spectrometry and collisional activation tandem mass spectrometry. J. Am. Soc. Mass Spectrom. 15, 1181–1190 (2004)

    Article  CAS  Google Scholar 

  27. Tureček, F.: Copper-biomolecule complexes in the gas phase. The ternary way. Mass Spectrom. Rev. 26, 563–582 (2007)

    Article  Google Scholar 

  28. Barlow, C.K., McFadyen, W.D., O’Hair, R.A.: Formation of cationic peptide radicals by gas-phase redox reactions with trivalent chromium, manganese, iron, and cobalt complexes. J. Am. Chem. Soc. 127, 6109–6115 (2005)

    Article  CAS  Google Scholar 

  29. Chu, I.K., Rodriquez, C.F., Lau, T.C., Hopkinson, A.C., Siu, K.M.: Molecular radical cations of oligopeptides. J. Phys. Chem. B 104, 3393–3397 (2000)

    Article  CAS  Google Scholar 

  30. Breuker, K., McLafferty, F.W.: Native electron capture dissociation for the structural characterization of noncovalent interactions in native cytochrome c**. Angew. Chem. 115, 5048–5052 (2003)

    Article  Google Scholar 

  31. Chan, W.C., White, P.D. (eds.): Fmoc solid phase peptide synthesis—a practical approach. Oxford University Press Inc, New York (2000)

    Google Scholar 

  32. Bleiholder, C., Paizs, B., Suhai, S.: Revising the proton affinity scale of the naturally occurring α-amino acids. J. Am. Soc. Mass Spectrom. 17, 1275–1281 (2006)

    Article  CAS  Google Scholar 

  33. Frisch, M.J., Trucks, G.W., Schlegel, H.B., Scuseria, G.E., Robb, M.A., Cheeseman, J.R., Scalmani, G., Barone, V., Mennucci, B., Petersson, G.A., Nakatsuji, H., Caricato, M., Li, X., Hratchian, H.P., Izmaylov, A.F., Bloino, J., Zheng, G., Sonnenberg, J.L., Hada, M., Ehara, M., Toyota, K., Fukuda, R., Hasegawa, J., Ishida, M., Nakajima, T., Honda, Y., Kitao, O., Nakai, H., Vreven, T., Montgomery Jr., J.A., Peralta, J.E., Ogliaro, F., Bearpark, M., Heyd, J.J., Brothers, E., Kudin, K.N., Staroverov, V.N., Kobayashi, R., Normand, J., Raghavachari, K., Rendell, A., Burant, J.C., Iyengar, S.S., Tomasi, J., Cossi, M., Rega, N., Millam, N.J., Klene, M., Knox, J.E., Cross, J.B., Bakken, V., Adamo, C., Jaramillo, J., Gomperts, R., Stratmann, R.E., Yazyev, O., Austin, A.J., Cammi, R., Pomelli, C., Ochterski, J.W., Martin, R.L., Morokuma, K., Zakrzewski, V.G., Voth, G.A., Salvador, P., Dannenberg, J.J., Dapprich, S., Daniels, A.D., Farkas, Ö., Foresman, J.B., Ortiz, J.V., Cioslowski, J., Fox, D.: Gaussian 09, revision A.01. Gaussian, Inc, Wallingford (2009)

    Google Scholar 

  34. MacKerell, A.D., Bashford, D., Bellott, M., Dunbrack, R.L., Evanseck, J.D., Field, M.J., Fischer, S., Gao, J., Guo, H., Ha, S., Joseph-McCarthy, D., Kuchnir, L., Kuczera, K., Lau, F.T.K., Mattos, C., Michnick, S., Ngo, T., Nguyen, D.T., Prodhom, B., Reiher, W.E., Roux, B., Schlenkrich, M., Smith, J.C., Stote, R., Straub, J., Watanabe, M., Wiorkiewicz-Kuczera, J., Yin, D., Karplus, M.: All-atom empirical potential for molecular modeling and dynamics studies of proteins. J. Phys. Chem. B 102, 3586–3616 (1998)

    Article  CAS  Google Scholar 

  35. Discovery Studio 2.5; Accelrys Inc.: San Diego (2009)

  36. Allen, J.M., Racine, A.H., Berman, A.M., Johnson, J.S., Bythell, B.J., Paizs, B., Glish, G.L.: Why are a3 ions rarely observed? J. Am. Soc. Mass Spectrom. 19, 1764–1770 (2008)

    Article  CAS  Google Scholar 

  37. Sinha, R.K., Erlekam, U., Bythell, B.J., Paizs, B., Maître, P.: Diagnosing the protonation site of b2 peptide fragment ions using IRMPD in the X–H (X = O, N, and C) stretching region. J. Am. Soc. Mass Spectrom. 22, 1645–1650 (2011)

    Article  CAS  Google Scholar 

  38. Tsaprailis, G., Nair, H., Zhong, W., Kuppannan, K., Futrell, J.H., Wysocki, V.H.: A mechanistic investigation of the enhanced cleavage at histidine in the gas-phase dissociation of protonated peptides. Anal. Chem. 76, 2083–2094 (2004)

    Article  CAS  Google Scholar 

  39. Farrugia, J.M., Taverner, T., O’Hair, R.A.: Side-chain involvement in the fragmentation reactions of the protonated methyl esters of histidine and its peptides. Int. J. Mass Spectrom. 209, 99–112 (2001)

    Article  CAS  Google Scholar 

  40. Barton, S.J., Whittaker, J.C.: Review of factors that influence the abundance of ions produced in a tandem mass spectrometer and statistical methods for discovering these factors. Mass Spectrom. Rev. 28, 177–187 (2009)

    Article  CAS  Google Scholar 

  41. Reid, G.E., Simpson, R.J., O’Hair, R.A.: Leaving group and gas phase neighboring group effects in the side chain losses from protonated serine and its derivatives. J. Am. Soc. Mass Spectrom. 11, 1047–1060 (2000)

    Article  CAS  Google Scholar 

  42. Neta, P., Pu, Q.L., Yang, X., Stein, S.E.: Consecutive neutral losses of H2O and C2H4O from N-terminal Thr–Thr and Thr–Ser in collision-induced dissociation of protonated peptides Position dependent water loss from single Thr or Ser. Int. J. Mass Spectrom. 267, 295–301 (2007)

    Article  CAS  Google Scholar 

  43. Serafin, S.V., Zhang, K., Aurelio, L., Hughes, A.B.., Morton, T.H.: Decomposition of protonated threonine, its stereoisomers, and its homologues in the gas phase: evidence for internal backside displacement. Org. Lett. 6, 1561–1564 (2004)

    Article  CAS  Google Scholar 

  44. Laskin, J., Yang, Z., Ng, C.M., Chu, I.K.: Fragmentation of α-radical cations of arginine-containing peptides. J. Am. Soc. Mass Spectrom. 21, 511–521 (2010)

    Article  CAS  Google Scholar 

  45. Singh, G.S., D’hooghe, M., Kimpe, N.D.: Synthesis and reactivity of C-heteroatom-substituted aziridines. Chem. Rev. 107, 2080–2135 (2007)

    Article  CAS  Google Scholar 

  46. Cooper, T.J., Talaty, E.R., Van Stipdonk, M.J.: Novel fragmentation pathway for CID of (bn-1+Cat)+ ions from model, metal cationized peptides. J. Am. Soc. Mass Spectrom. 16, 1305–1310 (2005)

    Article  CAS  Google Scholar 

  47. Katritzky, A.R., Wang, M., Wilkerson, C.R., Yang, H.: A novel approach to substituted 2H-azirines. J. Org. Chem. 68, 9105–9108 (2003)

    Article  CAS  Google Scholar 

Download references

Acknowledgment

The authors acknowledge support for this work by the National Natural Science Foundation of China (no. 21175056 and 51273080).

Author information

Authors and Affiliations

  1. College of Chemistry, Jilin University, Changchun, 130012, China

    Bing Wang, Huixin Wang, Zhonglin Wei, Xinhua Guo, Zhaohui Xiao & Zhoufang Zeng

  2. The State Engineering Laboratory of AIDS Vaccine, Jilin University, Changchun, 130012, China

    Jiayi Yu & Wei Kong

Authors
  1. Bing Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jiayi Yu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Huixin Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Zhonglin Wei
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Xinhua Guo
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Zhaohui Xiao
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Zhoufang Zeng
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Wei Kong
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Xinhua Guo or Wei Kong.

Additional information

Bing Wang and Jiayi Yu contributed equally to this work.

Electronic supplementary material

Below is the link to the electronic supplementary material.

ESM 1

(DOC 2440 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Wang, B., Yu, J., Wang, H. et al. Investigation of bn-44 Peptide Fragments Using High Resolution Mass Spectrometry and Isotope Labeling. J. Am. Soc. Mass Spectrom. 25, 2116–2124 (2014). https://doi.org/10.1007/s13361-014-0994-9

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s13361-014-0994-9

Keywords

Search

Navigation