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Applying Arginylation for Bottom-Up Proteomics

  • H. Alexander EbhardtEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1337)

Abstract

Arginylation is an enzymatic reaction in which arginyl-tRNA protein transferase 1 (ATE1, EC 2.3.2.8) conjugates a single arginyl moiety from aminoacylated tRNAArg onto a target polypeptide. We established arginylation for in vitro labeling of peptides with N-terminal acidic amino acids. Consistent with prior knowledge, arginylated peptides flanked by basic amino acids result in rich redundant MS/MS fragment spectra using various precursor fragmentation modes. Arginylation carried out by ATE1 is a fast method for labeling peptides. Sequence-specific proteolytic digest of proteins is best carried out using a double digest of proteins by Lys-C and Asp-N to generate peptides with a basic amino acid on the C-terminus and an acidic amino acid on the N-terminus. Under these conditions, arginylation is specific for N-terminal acidic amino acids and results in a near 2× sequence coverage in the MS/MS spectrum are achieved.

Key words

Selected reaction monitoring (SRM) Multiple reaction monitoring (MRM) Mass spectrometry Arginylation 

Notes

Acknowledgments

This work was supported by a FP7 Marie Curie International Incoming Fellowship to HAE.

References

  1. 1.
    Kebarle P (2000) A brief overview of the present status of the mechanisms involved in electrospray mass spectrometry. J Mass Spectrom 35(7):804–817. doi: 10.1002/1096-9888(200007)35:7<804::AID-JMS22>3.0.CO;2-Q CrossRefPubMedGoogle Scholar
  2. 2.
    Dole M, Mack LL, Hines RL (1968) Molecular beams of macroions. J Chem Phys 49(5):2240–2249. doi: 10.1063/1.1670391 CrossRefGoogle Scholar
  3. 3.
    Fenn JB, Mann M, Meng CK, Wong SF, Whitehouse CM (1990) Electrospray ionization? Principles and practice. Mass Spectrom Rev 9(1):37–70CrossRefGoogle Scholar
  4. 4.
    Nguyen S, Fenn JB (2007) Gas-phase ions of solute species from charged droplets of solutions. Proc Natl Acad Sci U S A 104(4):1111–1117. doi: 10.1073/pnas.0609969104 PubMedCentralCrossRefPubMedGoogle Scholar
  5. 5.
    Carr SR, Cassady CJ (1997) Reactivity and gas-phase acidity determinations of small peptide ions consisting of 11 to 14 amino acid residues. J Mass Spectrom 32(9):959–967. doi: 10.1002/(SICI)1096-9888(199709)32:9<959::AID-JMS552>3.0.CO;2-5 CrossRefPubMedGoogle Scholar
  6. 6.
    Carabetta VJ, Li T, Shakya A, Greco TM, Cristea IM (2010) Integrating Lys-N proteolysis and N-terminal guanidination for improved fragmentation and relative quantification of singly-charged ions. J Am Soc Mass Spectrom 21(6):1050–1060. doi: 10.1016/j.jasms.2010.02.004 PubMedCentralCrossRefPubMedGoogle Scholar
  7. 7.
    Boersema PJ, Taouatas N, Altelaar AF, Gouw JW, Ross PL, Pappin DJ, Heck AJ, Mohammed S (2009) Straightforward and de novo peptide sequencing by MALDI-MS/MS using a Lys-N metalloendopeptidase. Mol Cell Proteomics 8(4):650–660. doi: 10.1074/mcp.M800249-MCP200 PubMedCentralCrossRefPubMedGoogle Scholar
  8. 8.
    Somogyi A, Wysocki VH, Mayer I (1994) The effect of protonation site on bond strengths in simple peptides: application of ab initio and modified neglect of differential overlap bond orders and modified neglect of differential overlap energy partitioning. J Am Soc Mass Spectrom 5(8):704–717. doi: 10.1016/1044-0305(94)80002-2 CrossRefPubMedGoogle Scholar
  9. 9.
    Dongre AR, Somogyi A, Wysocki VH (1996) Surface-induced dissociation: an effective tool to probe structure, energetics and fragmentation mechanisms of protonated peptides. J Mass Spectrom 31(4):339–350. doi: 10.1002/(SICI)1096-9888(199604)31:4<339::AID-JMS322>3.0.CO;2-L CrossRefPubMedGoogle Scholar
  10. 10.
    Vaisar T, Urban J (1996) Probing the proline effect in CID of protonated peptides. J Mass Spectrom 31(10):1185–1187. doi: 10.1002/(SICI)1096-9888(199610)31:10<1185::AID-JMS396>3.0.CO;2-Q CrossRefPubMedGoogle Scholar
  11. 11.
    Tsaprailis G, Nair H, Zhong W, Kuppannan K, Futrell JH, Wysocki VH (2004) A mechanistic investigation of the enhanced cleavage at histidine in the gas-phase dissociation of protonated peptides. Anal Chem 76(7):2083–2094. doi: 10.1021/ac034971j CrossRefPubMedGoogle Scholar
  12. 12.
    Seidler J, Zinn N, Boehm ME, Lehmann WD (2010) De novo sequencing of peptides by MS/MS. Proteomics 10(4):634–649. doi: 10.1002/pmic.200900459 CrossRefPubMedGoogle Scholar
  13. 13.
    Domon B, Aebersold R (2006) Challenges and opportunities in proteomics data analysis. Mol Cell Proteomics 5(10):1921–1926. doi: 10.1074/mcp.R600012-MCP200 CrossRefPubMedGoogle Scholar
  14. 14.
    Dancik V, Addona TA, Clauser KR, Vath JE, Pevzner PA (1999) De novo peptide sequencing via tandem mass spectrometry. J Comput Biol 6(3–4):327–342. doi: 10.1089/106652799318300 CrossRefPubMedGoogle Scholar
  15. 15.
    Frank AM, Savitski MM, Nielsen ML, Zubarev RA, Pevzner PA (2007) De novo peptide sequencing and identification with precision mass spectrometry. J Proteome Res 6(1):114–123. doi: 10.1021/pr060271u PubMedCentralCrossRefPubMedGoogle Scholar
  16. 16.
    Mann M, Wilm M (1994) Error-tolerant identification of peptides in sequence databases by peptide sequence tags. Anal Chem 66(24):4390–4399CrossRefPubMedGoogle Scholar
  17. 17.
    Kapp EA, Schutz F, Connolly LM, Chakel JA, Meza JE, Miller CA, Fenyo D, Eng JK, Adkins JN, Omenn GS, Simpson RJ (2005) An evaluation, comparison, and accurate benchmarking of several publicly available MS/MS search algorithms: sensitivity and specificity analysis. Proteomics 5(13):3475–3490. doi: 10.1002/pmic.200500126 CrossRefPubMedGoogle Scholar
  18. 18.
    Creasy DM, Cottrell JS (2002) Error tolerant searching of uninterpreted tandem mass spectrometry data. Proteomics 2(10):1426–1434. doi: 10.1002/1615-9861(200210)2:10<1426::AID-PROT1426>3.0.CO;2-5 CrossRefPubMedGoogle Scholar
  19. 19.
    McCormack AL, Schieltz DM, Goode B, Yang S, Barnes G, Drubin D, Yates JR III (1997) Direct analysis and identification of proteins in mixtures by LC/MS/MS and database searching at the low-femtomole level. Anal Chem 69(4):767–776CrossRefPubMedGoogle Scholar
  20. 20.
    Eng JK, McCormack AL, Yates JR (1994) An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database. J Am Soc Mass Spectrom 5(11):976–989. doi: 10.1016/1044-0305(94)80016-2 CrossRefPubMedGoogle Scholar
  21. 21.
    Michalski A, Cox J, Mann M (2011) More than 100,000 detectable peptide species elute in single shotgun proteomics runs but the majority is inaccessible to data-dependent LC-MS/MS. J Proteome Res 10(4):1785–1793. doi: 10.1021/pr101060v CrossRefPubMedGoogle Scholar
  22. 22.
    Balzi E, Choder M, Chen WN, Varshavsky A, Goffeau A (1990) Cloning and functional analysis of the arginyl-tRNA-protein transferase gene ATE1 of Saccharomyces cerevisiae. J Biol Chem 265(13):7464–7471PubMedGoogle Scholar
  23. 23.
    Ebhardt HA, Nan J, Chaulk SG, Fahlman RP, Aebersold R (2014) Enzymatic generation of peptides flanked by basic amino acids to obtain MS/MS spectra with 2× sequence coverage. Rapid Commun Mass Spectrom 28(24):2735–2743. doi: 10.1002/rcm.7069 PubMedCentralCrossRefPubMedGoogle Scholar
  24. 24.
    Shi PY, Weiner AM, Maizels N (1998) A top-half tDNA minihelix is a good substrate for the eubacterial CCA-adding enzyme. RNA (New York, NY) 4(3):276–284Google Scholar
  25. 25.
    Ebhardt HA, Xu Z, Fung AW, Fahlman RP (2009) Quantification of the post-translational addition of amino acids to proteins by MALDI-TOF mass spectrometry. Anal Chem 81(5):1937–1943CrossRefPubMedGoogle Scholar
  26. 26.
    Fung AW, Ebhardt HA, Abeysundara H, Moore J, Xu Z, Fahlman RP (2011) An alternative mechanism for the catalysis of peptide bond formation by L/F transferase: substrate binding and orientation. J Mol Biol 409(4):617–629. doi: 10.1016/j.jmb.2011.04.033, S0022-2836(11)00448-7 [pii]CrossRefPubMedGoogle Scholar
  27. 27.
    Juhling F, Morl M, Hartmann RK, Sprinzl M, Stadler PF, Putz J (2009) tRNAdb 2009: compilation of tRNA sequences and tRNA genes. Nucleic Acids Res 37(Database issue):D159–D162PubMedCentralCrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.Institute of Molecular Systems BiologyEidgenössische Technische Hochschule (ETH) ZürichZürichSwitzerland

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