Top-Down Analysis of Proteins in Low Charge States

  • Aarti Bashyal
  • James D. Sanders
  • Dustin D. Holden
  • Jennifer S. BrodbeltEmail author
Research Article


The impact of charging methods on the dissociation behavior of intact proteins in low charge states is investigated using HCD and 193 nm UVPD. Low charge states are produced for seven different proteins using the following four different methods: (1) proton transfer reactions of ions in high charge states generated from conventional denaturing solutions; (2) ESI of proteins in solutions of high ionic strength to enhance retention of folded native-like conformations; (3) ESI of proteins in high pH solutions to limit protonation; and (4) ESI of carbamylated proteins. Comparison of sequence coverages, degree of preferential cleavages, and types and distribution of fragment ions reveals a number of differences in the fragmentation patterns depending on the method used to generate the ions. More notable differences in these metrics are observed upon HCD than upon UVPD. The fragmentation caused by HCD is influenced more significantly by the presence/absence of mobile protons, a factor that modulates the degree of preferential cleavages and net sequence coverages. Carbamylation of the lysines and the N-terminus of the proteins alters the proton mobility by reducing the number of proton-sequestering, highly basic sites as evidenced by decreased preferential fragmentation C-terminal to Asp or N-terminal to Pro upon HCD. UVPD is less dependent on the method used to generate the low charge states and favors non-specific fragmentation, an outcome which is important for obtaining high sequence coverage of intact proteins.


Top-down Ultraviolet photodissociation Protein Low charge state 



We appreciate the donation of proteins from Dr. Kevin Dalby (calmodulin) and Dr. Lauren Webb (Staph nuclease). Funding from the NIH (R01GM121714) and the Robert A. Welch Foundation (F-1155) is acknowledged.

Supplementary material

13361_2019_2146_MOESM1_ESM.pdf (2.5 mb)
ESM 1 (PDF 2605 kb)


  1. 1.
    Toby, T.K., Fornelli, L., Kelleher, N.L.: Progress in top-down proteomics and the analysis of proteoforms. Annu. Rev. Anal. Chem. 9, 499–519 (2016)CrossRefGoogle Scholar
  2. 2.
    Catherman, A.D., Skinner, O.S., Kelleher, N.L.: Top down proteomics: facts and perspectives. Biochem. Biophys. Res. Commun. 445, 683–693 (2014)CrossRefGoogle Scholar
  3. 3.
    Smith, L.M., Kelleher, N.L., Proteomics, T.C. for T.D., Linial, M., Goodlett, D., Langridge-Smith, P., Goo, Y.A., Safford, G., Bonilla*, L., Kruppa, G., Zubarev, R., Rontree, J., Chamot-Rooke, J., Garavelli, J., Heck, A., Loo, J., Penque, D., Hornshaw, M., Hendrickson, C., Pasa-Tolic, L., Borchers, C., Chan, D., Young*, N., Agar, J., Masselon, C., Gross*, M., McLafferty, F., Tsybin, Y., Ge, Y., Sanders*, I., Langridge, J., Whitelegge*, J., Marshall, A: Proteoform: a single term describing protein complexity. Nat. Methods. 10, 186–187 (2013)CrossRefGoogle Scholar
  4. 4.
    Durbin, K.R., Fornelli, L., Fellers, R.T., Doubleday, P.F., Narita, M., Kelleher, N.L.: Quantitation and identification of thousands of human proteoforms below 30 kDa. J. Proteome Res. 15, 976–982 (2016)CrossRefGoogle Scholar
  5. 5.
    Reid, G.E., Wu, J., Chrisman, P.A., Wells, J.M., McLuckey, S.A.: Charge-state-dependent sequence analysis of protonated ubiquitin ions via ion trap tandem mass spectrometry. Anal. Chem. 73, 3274–3281 (2001)CrossRefGoogle Scholar
  6. 6.
    Chanthamontri, C., Liu, J., McLuckey, S.A.: Charge state dependent fragmentation of gaseous α-synuclein cations via ion trap and beam-type collisional activation. Int. J. Mass Spectrom. 283, 9–16 (2009)CrossRefGoogle Scholar
  7. 7.
    Dongré, 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)CrossRefGoogle Scholar
  8. 8.
    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)CrossRefGoogle Scholar
  9. 9.
    Haverland, N.A., Skinner, O.S., Fellers, R.T., Tariq, A.A., Early, B.P., LeDuc, R.D., Fornelli, L., Compton, P.D., Kelleher, N.L.: Defining gas-phase fragmentation propensities of intact proteins during native top-down mass spectrometry. J. Am. Soc. Mass Spectrom. 28, 1203–1215 (2017)CrossRefGoogle Scholar
  10. 10.
    Cobb, J.S., Easterling, M.L., Agar, J.N.: Structural characterization of intact proteins is enhanced by prevalent fragmentation pathways rarely observed for peptides. J. Am. Soc. Mass Spectrom. 21, 949–959 (2010)CrossRefGoogle Scholar
  11. 11.
    Morrison, L.J., Brodbelt, J.S.: Charge site assignment in native proteins by ultraviolet photodissociation (UVPD) mass spectrometry. Analyst. 141, 166–176 (2015)CrossRefGoogle Scholar
  12. 12.
    Olsen, J.V., Macek, B., Lange, O., Makarov, A., Horning, S., Mann, M.: Higher-energy C-trap dissociation for peptide modification analysis. Nat. Methods. 4, 709–712 (2007)CrossRefGoogle Scholar
  13. 13.
    Brodbelt, J.S.: Ion activation methods for peptides and proteins. Anal. Chem. 88, 30–51 (2016)CrossRefGoogle Scholar
  14. 14.
    Min-Sik, K., Akhilesh, P.: Electron transfer dissociation mass spectrometry in proteomics. Proteomics. 12, 530–542 (2012)CrossRefGoogle Scholar
  15. 15.
    Syka, J.E.P., Coon, J.J., Schroeder, M.J., Shabanowitz, J., Hunt, D.F.: Peptide and protein sequence analysis by electron transfer dissociation mass spectrometry. Proc. Natl. Acad. Sci. 101, 9528–9533 (2004)CrossRefGoogle Scholar
  16. 16.
    Riley, N.M., Coon, J.J.: The role of electron transfer dissociation in modern proteomics. Anal. Chem. 90, 40–64 (2018)CrossRefGoogle Scholar
  17. 17.
    Cannon, J.R., Martinez-Fonts, K., Robotham, S.A., Matouschek, A., Brodbelt, J.S.: Top-down 193-nm ultraviolet photodissociation mass spectrometry for simultaneous determination of polyubiquitin chain length and topology. Anal. Chem. 87, 1812–1820 (2015)CrossRefGoogle Scholar
  18. 18.
    Greer, S.M., Holden, D.D., Fellers, R., Kelleher, N.L., Brodbelt, J.S.: Modulation of protein fragmentation through carbamylation of primary amines. J. Am. Soc. Mass Spectrom. 28, 1587–1599 (2017)CrossRefGoogle Scholar
  19. 19.
    Shaw, J.B., Li, W., Holden, D.D., Zhang, Y., Griep-Raming, J., Fellers, R.T., Early, B.P., Thomas, P.M., Kelleher, N.L., Brodbelt, J.S.: Complete protein characterization using top-down mass spectrometry and ultraviolet photodissociation. J. Am. Chem. Soc. 135, 12646–12651 (2013)CrossRefGoogle Scholar
  20. 20.
    Cannon, J.R., Cammarata, M.B., Robotham, S.A., Cotham, V.C., Shaw, J.B., Fellers, R.T., Early, B.P., Thomas, P.M., Kelleher, N.L., Brodbelt, J.S.: Ultraviolet photodissociation for characterization of whole proteins on a chromatographic time scale. Anal. Chem. 86, 2185–2192 (2014)CrossRefGoogle Scholar
  21. 21.
    Greer, S.M., Brodbelt, J.S.: Top-down characterization of heavily modified histones using 193 nm ultraviolet photodissociation mass spectrometry. J. Proteome Res. 17, 1138–1145 (2018)CrossRefGoogle Scholar
  22. 22.
    Julian, R.R.: The mechanism behind top-down UVPD experiments: making sense of apparent contradictions. J. Am. Soc. Mass Spectrom. 28, 1823–1826 (2017)CrossRefGoogle Scholar
  23. 23.
    Susa, A.C., Xia, Z., Tang, H.Y.H., Tainer, J.A., Williams, E.R.: Charging of proteins in native mass spectrometry. J. Am. Soc. Mass Spectrom. 28, 332–340 (2017)CrossRefGoogle Scholar
  24. 24.
    Konermann, L.: Addressing a common misconception: ammonium acetate as neutral pH “buffer” for native electrospray mass spectrometry. J. Am. Soc. Mass Spectrom. 28, 1827–1835 (2017)CrossRefGoogle Scholar
  25. 25.
    Holden, D.D., McGee, W.M., Brodbelt, J.S.: Integration of ultraviolet photodissociation with proton transfer reactions and ion parking for analysis of intact proteins. Anal. Chem. 88, 1008–1016 (2016)CrossRefGoogle Scholar
  26. 26.
    Iavarone, A.T., Jurchen, J.C., Williams, E.R.: Effects of solvent on the maximum charge state and charge state distribution of protein ions produced by electrospray ionization. J. Am. Soc. Mass Spectrom. 11, 976–985 (2000)CrossRefGoogle Scholar
  27. 27.
    Krusemark, C.J., Frey, B.L., Belshaw, P.J., Smith, L.M.: Modifying the charge state distribution of proteins in electrospray ionization mass spectrometry by chemical derivatization. J. Am. Soc. Mass Spectrom. 20, 1617–1625 (2009)CrossRefGoogle Scholar
  28. 28.
    Iavarone, A.T., Williams, E.R.: Collisionally activated dissociation of supercharged proteins formed by electrospray ionization. Anal. Chem. 75, 4525–4533 (2003)CrossRefGoogle Scholar
  29. 29.
    Teo, C.A., Donald, W.A.: Solution additives for supercharging proteins beyond the theoretical maximum proton-transfer limit in electrospray ionization mass spectrometry. Anal. Chem. 86, 4455–4462 (2014)CrossRefGoogle Scholar
  30. 30.
    Wysocki, V.H., George, T., Smith Lori, L., Breci, L.A.: Mobile and localized protons: a framework for understanding peptide dissociation. J. Mass Spectrom. 35, 1399–1406 (2001)CrossRefGoogle Scholar
  31. 31.
    Pitteri, S.J., Reid, G.E., McLuckey, S.A.: Affecting proton mobility in activated peptide and whole protein ions via lysine guanidination. J. Proteome Res. 3, 46–54 (2004)CrossRefGoogle Scholar
  32. 32.
    Lee, K., Alphonse, S., Piserchio, A., Tavares, C.D.J., Giles, D.H., Wellmann, R.M., Dalby, K.N., Ghose, R.: Structural basis for the recognition of eukaryotic elongation factor 2 kinase by calmodulin. Struct. Lond. Engl. 1993(24), 1441–1451 (2016)Google Scholar
  33. 33.
    Greer, S.M., Cannon, J.R., Brodbelt, J.S.: Improvement of shotgun proteomics in the negative mode by carbamylation of peptides and ultraviolet photodissociation mass spectrometry. Anal. Chem. 86, 12285–12290 (2014)CrossRefGoogle Scholar
  34. 34.
    Sanders, J.D., Grinfeld, D., Aizikov, K., Makarov, A., Holden, D.D., Brodbelt, J.S.: Determination of collision cross-sections of protein ions in an orbitrap mass analyzer. Anal. Chem. 90, 5896–5902 (2018)CrossRefGoogle Scholar
  35. 35.
    Fellers, R.T., Greer, J.B., Early, B.P., Yu, X., LeDuc, R.D., Kelleher, N.L., Thomas, P.M.: ProSight lite: graphical software to analyze top-down mass spectrometry data. Proteomics. 15, 1235–1238 (2015)CrossRefGoogle Scholar
  36. 36.
    Rosenberg, J., Parker, W.R., Cammarata, M.B., Brodbelt, J.S.: UV-POSIT: Web-based tools for rapid and facile structural interpretation of ultraviolet photodissociation (UVPD) mass spectra. J. Am. Soc. Mass Spectrom. 29, 1323–1326 (2018)Google Scholar
  37. 37.
    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)CrossRefGoogle Scholar
  38. 38.
    Morrison, L.J., Rosenberg, J.A., Singleton, J.P., Brodbelt, J.S.: Statistical examination of the a and a+1 fragment ions from 193 nm UVPD reveals local hydrogen bonding interactions. J. Am. Soc. Mass Spectrom. 27, 1443–1453 (2016)CrossRefGoogle Scholar
  39. 39.
    Harper, B., Neumann, E.K., Stow, S.M., May, J.C., McLean, J.A., Solouki, T.: Determination of ion mobility collision cross sections for unresolved isomeric mixtures using tandem mass spectrometry and chemometric deconvolution. Anal. Chim. Acta. 939, 64–72 (2016)CrossRefGoogle Scholar
  40. 40.
    Shi, H., Atlasevich, N., Merenbloom, S.I., Clemmer, D.E.: Solution dependence of the collisional activation of ubiquitin [M + 7H]7+ ions. J. Am. Soc. Mass Spectrom. 25, 2000–2008 (2014)CrossRefGoogle Scholar
  41. 41.
    Shi, H., Pierson, N.A., Valentine, S.J., Clemmer, D.E.: Conformation types of ubiquitin [M + 8H]8+ ions from water:methanol solutions: evidence for the N and A states in aqueous solution. J. Phys. Chem. B. 116, 3344–3352 (2012)CrossRefGoogle Scholar
  42. 42.
    Valentine, S.J., Counterman, A.E., Clemmer, D.E.: Conformer-dependent proton-transfer reactions of ubiquitin ions. J. Am. Soc. Mass Spectrom. 8, 954–961 (1997)CrossRefGoogle Scholar
  43. 43.
    Laszlo, K.J., Bush, M.F.: Interpreting the collision cross sections of native-like protein ions: insights from cation-to-anion proton-transfer reactions. Anal. Chem. 89, 7607–7614 (2017)CrossRefGoogle Scholar
  44. 44.
    Bush, M.F., Hall, Z., Giles, K., Hoyes, J., Robinson, C.V., Ruotolo, B.T.: Collision cross sections of proteins and their complexes: a calibration framework and database for gas-phase structural biology. Anal. Chem. 82, 9557–9565 (2010)CrossRefGoogle Scholar
  45. 45.
    Salbo, R., Bush, M.F., Naver, H., Campuzano, I., Robinson, C.V., Pettersson, I., Jørgensen, T.J.D., Haselmann, K.F.: Traveling-wave ion mobility mass spectrometry of protein complexes: accurate calibrated collision cross-sections of human insulin oligomers. Rapid Commun. Mass Spectrom. 26, 1181–1193 (2012)CrossRefGoogle Scholar
  46. 46.
    Lermyte, F., Łącki, M.K., Valkenborg, D., Gambin, A., Sobott, F.: Conformational space and stability of ETD charge reduction products of ubiquitin. J. Am. Soc. Mass Spectrom. 28, 69–76 (2017)CrossRefGoogle Scholar
  47. 47.
    Laszlo, K.J., Munger, E.B., Bush, M.F.: Folding of protein ions in the gas phase after cation-to-anion proton-transfer reactions. J. Am. Chem. Soc. 138, 9581–9588 (2016)CrossRefGoogle Scholar
  48. 48.
    Laszlo, K.J., Buckner, J.H., Munger, E.B., Bush, M.F.: Native-like and denatured cytochrome c ions yield cation-to-anion proton transfer reaction products with similar collision cross-sections. J. Am. Soc. Mass Spectrom. 28, 1382–1391 (2017)CrossRefGoogle Scholar
  49. 49.
    Jhingree, J.R., Beveridge, R., Dickinson, E.R., Williams, J.P., Brown, J.M., Bellina, B., Barran, P.E.: Electron transfer with no dissociation ion mobility–mass spectrometry (ETnoD IM-MS). The effect of charge reduction on protein conformation. Int. J. Mass Spectrom. 413, 43–51 (2017)CrossRefGoogle Scholar
  50. 50.
    Mosier, P.D., Counterman, A.E., Jurs, P.C., Clemmer, D.E.: Prediction of peptide ion collision cross sections from topological molecular structure and amino acid parameters. Anal. Chem. 74, 1360–1370 (2002)CrossRefGoogle Scholar
  51. 51.
    Loo, R.R.O., Loo, J.A.: Salt bridge rearrangement (SaBRe) explains the dissociation behavior of noncovalent complexes. J. Am. Soc. Mass Spectrom. 27, 975–990 (2016)CrossRefGoogle Scholar

Copyright information

© American Society for Mass Spectrometry 2019

Authors and Affiliations

  1. 1.Department of ChemistryThe University of Texas at AustinAustinUSA

Personalised recommendations