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Size-dependent hydrogen atom attachment to gasphase hydrogen-deficient polypeptide radical cations Luciano di Stefano, Dimitris Papanastasiou, and Roman A. Zubarev J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.7b10318 • Publication Date (Web): 02 Jan 2018 Downloaded from http://pubs.acs.org on January 2, 2018
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Journal of the American Chemical Society
Size-dependent hydrogen atom attachment to gas-phase hydrogendeficient polypeptide radical cations Luciano H. Di Stefano,†,§ Dimitris Papanastasiou,‡,§ Roman A. Zubarev*,† †
Division of Physiological Chemistry I, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Scheelesväg 2, S-171 77 Stockholm, Sweden
‡
Fasmatech Science & Technology, Lefkippos TESPA, Demokritos NCSR, PatriarchouGregoriou&Neapoleos, 153-10 Agia Paraskevi, Athens, Greece
Supporting Information: see Supplementary materials ABSTRACT: Despite significant affinity to carbonyl oxygens, thermal hydrogen atoms attach to unmodified polypeptides at a very low rate, while the hydrogen-hydrogen exchange rate is high. Here, using the novel omnitrap set-up, we found that attachment to polypeptides is much more facile when radical site is already present, but the rate decreases for larger radical ions. The likely explanation is the intramolecular hydrogen atom rearrangement in hydrogen-deficient radicals to a more stable or less accessible site.
Tandem mass spectrometry (MS/MS) identifies known sequences of polypeptides or sequences them de novo by fragmenting multiply protonated molecules in the gas phase and analyzing the fragment masses. For successful analysis, as many as possible inter-residue bonds need to be cleaved with similar probabilities, preferably just one bond cleaved per molecule. Homogeneous backbone cleavage can be achieved by converting multiply protonated molecules to radical cations. •
Polypeptide radicals [M + nH]m+ come in two kinds: hydrogen-deficient (nm) radicals, with vastly different fragmentation behavior of these two classes.1-5 Hydrogen-deficient radicals are relatively easy to obtain, e.g., by ionization of (poly)-protonated polypeptides by UV photons or >10 eV electrons.3 However, their fragmentation often adds limited sequence information to that available from MS/MS of evenelectron precursor ions. In contrast, hydrogen-abundant radicals yield wealth of structural information, being the crucial intermediates in electron capture dissociation (ECD)1 and electron transfer dissociation (ETD)6. Thus conversion of polypeptide molecules or their hydrogen-deficient radicals into hydrogen-abundant radicals may help improving polypeptide sequencing with MS/MS.3 Early attempts to achieve direct attachment of thermal hydrogen atoms (≤0.1 eV) to gas-phase protonated polypeptides were unsuccessful.7-8 No capture was observed even in the presence of disulfide bonds that are believed to be more susceptible to hydrogen atom attack9 than carbonyl oxygens, for which the capture process is ≈0.6 eV exothermic8. At the same time, multiple hydrogen atom attachment was observed to C60 radicals7, which can be formally classified as hydrogen-deficient species. An important question is whether hydrogen-deficient polypeptide radicals are capable of capturing thermal hydrogen atoms, similar to their C60+• counterparts. Recently, it has become possi-
ble to address this issue experimentally using the omnitrap10 technology. An omnitrap is a string of several (9 in our implementation) serially connected linear ion traps, with at least some of the traps enabling diverse ion manipulation techniques. These techniques include ion activation using electrons with energy up to 1000 eV as well as thermal (≈1300 oC) hydrogen atoms. The omnitrap is connected to a high-resolution mass analyzer, in our case an Orbitrap. It is thus possible to protonate polypeptides by electrospray, isolate precursor ions in the omnitrap, meta-ionize them with >10 eV electrons to hydrogen-deficient radical cations, irradiate these with thermal hydrogen atoms, and observe the products in the Orbitrap analyzer. The target reactions were: •
[M + nH]n+ + e-fast → [M + nH](n+1)+ + 2e[M + nH]
(n+1)+•
+ H⋅ → [M + (n+1)H]
(1a)
(n+1)+
(1b)
Combined, (1a-b) amount to super-protonation in the gas phase of multiply protonated polypeptide molecules, a novel reaction yet to be observed experimentally. Achieving it would allow one to easily proceed to the next step, which is electron capture leading to hydrogen-abundant radicals: •
[M + (n+1)H](n+1)+ + e-slow → [M + (n+1)H]n+ *
(2)
Note that here, unlike in ECD and ETD, the hydrogen-abundant radicals are in the same charge state as the original precursors in (1a). Thus, (1-2) would give an ECD-type abundant sequence information without charge reduction and thus without sensitivity loss, an important goal in tandem mass spectrometry. Indeed, the absence of charge reduction would make MALDI-produced molecular species, typically singly-charged, amenable to radicalinduced dissociation, facilitating the use of MS/MS sequencing in e.g. MALDI imaging. In ECD/ETD of doubly-charged ions, one of the two complementary fragments is neutral, which leads to significant loss of sequence information; this problem would be eliminated in (1-2). Against expectations, we found the rate of (1b) to be very low for 4+⋅ cations of insulin B-chain (3.4 kDa), with hydrogen atom attachment barely detectable after 10 s of irradiation. At the same time, replacement of hydrogen gas with deuterium gave facile H/D exchange on millisecond time scale (data not shown), indicating that hydrogen atoms reached the polycationic radicals but did not get absorbed by the latter. Cytochrome C protonated molecules that due to the Fe(III) heme also have an unpaired electron and thus are radicals, did not attach hydrogen atoms either.
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The lack of H⋅ addition to polypeptide molecular radicals was even more puzzling since not only hydrogen atoms readily attach to C60+• radicals,7-8 but also hydrogen-deficient ECD products, z⋅ ions, rapidly appropriate hydrogen atoms from their complementary even-electron c´ counterparts.11 To investigate this contradiction in more detail, ECD of cytochrome C ions was performed in the omnitrap, with subsequent irradiation of the products by H⋅ atoms. Here, three attachment-related phenomena were observed (Figure 1): A) hydrogen atoms readily attach to z⋅ions; B) the
. Figure 1. H. addition to ECD fragments of cytochrome C: (A) radical ion, (B) heme group (+0.92 ±0.01 Da), (C) size-dependent shift in average molecular mass M of the radical z. ions and its absence for even-electron c´ and y ions.
. Figure 2. H. addition to ECD fragments of ubiquitin: (A) to radical ion; (B) to even-electron ion (no attachment); (C) size-dependent shift in average molecular mass M of the radical z. ions and its absence for even-electron c´ and y ions.
covalently bound heme group liberated in the ECD process9 absorbed H⋅ even faster, with attachment of up to four atoms; C) as expected, H⋅ atoms did not attach to even-electron c´ and y ions. Importantly, the attachment rate declined with the size of z⋅ ions, from on average ≈0.5 H atoms per ion for 5-10 residues large z⋅ ions, to half of that for large radical species comprising 25-35 residues. Similar experiments with ubiquitin gave nearly identical results (Figure 2). The likely explanation for the observed phenomena is the facile intramolecular hydrogen atom rearrangement in hydrogendeficient radicals immediately upon formation.12 Such rearrangement can lead to a more stable radical site, such as carboxylic acid and aromatic groups, and/or a less accessible site, e.g., shielded by elements of tertiary structure. As known from intramolecular hydrogen atom rearrangement between the complementary ECD fragments, more stable radicals are less prone to attach hydrogen atom.11 Also, in larger radicals the rate of hydrogen transfer must be faster than in small ones due to parallel existence of several transfer routes and a larger number of potential low-energy radical sites. At the same time, the larger surface area and more evolved tertiary structure should make it less probable that the incoming hydrogen atoms reach the radical site. All these factors may account for the observed decrease in the attachment rate of hydrogen atoms in larger hydrogen-deficient radicals. Recently, the Tanaka team have provided several intriguing reports on hydrogen atom interactions with MALDI-produced protonated peptides in the gas phase.13-14 With ≈0.15 eV thermal hydrogens, they reported H attachment to the carbonyl oxygen and H abstraction from the peptide backbone, which gave hydrogen-abundant and hydrogen-deficient radicals, respectively. Our experiments performed on larger molecules are mostly in agreement with these results. Tanaka team have found that the attachment rate seems to depend upon the energy of incoming hydrogen atoms, with higher energies being advantageous.14 With 10-30 s long irradiation times, they have managed to induce fragmentation of the polypeptides that was similar to ECD/ETD fragmentation but did not involve charge reduction. These efforts underline the importance of such a process for tandem mass spectrometry. Summarizing, here we confirmed, for the first time, the much higher attachment rate of hydrogen atoms to hydrogen-deficient radical cations compared to protonated molecules. Yet the rapidly declining cross section of this reaction with the size of the polypeptide radicals makes it difficult to implement the superprotonation reactions (1-2) for larger proteins, which would be of particular interest. A possible way to solve this problem is to increase the kinetic energy of hydrogen atoms above 0.15 eV.14 Alternatively, electronic excitation of either hydrogen atoms or the target protein radicals also may increase the attachment rate.15 As a final note, an increase in the polypeptide protonation state can occur in a single or sequential ion-ion reaction involving in the final step a (multiply) protonated molecule as a protonation agent.16 Such reactions are easier achieved via disruption of a saltbridge (H+)..(OH-)..(H+) with subsequent anion neutralization rather than via proton capture by a neutral site in a protonated molecule. Since most gas phase proteins are zwitterions in their “native” charge state,17-19 this pathway may hold a largely untapped potential for achieving meta-protonation.
ASSOCIATED CONTENT Supporting Information: Schematic diagram of the omni-
trap cross-section.
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AUTHOR INFORMATION Corresponding Author *
[email protected] (11)
Author Contributions §
(12)
LdS and DP contributed equally.
(13)
Notes The authors declare no competing financial interests.
(14)
ACKNOWLEDGMENT The authors gratefully acknowledge technical help of the Fasmatech R&D team in the development of the omnitrap platform. (15) (16) (17)
REFERENCES (1) (2) (3)
(4) (5) (6) (7) (8) (9)
Zubarev, R. A.; Kelleher, N. L.; McLafferty, F. W. J. Am. Chem. Soc. 1998, 120, 3265. Zubarev, R. A. Mass Spectrom. Rev.2003, 22, 57. Zubarev, R. A. Peptide Radical Cations: Gender Determines Dissociation Chemistry, in Advances in Mass Spectrometry, v. 19, pp. 43-46. IntAcadPublCo., Tokyo, Japan, 2013. Moore, B. N.; Ly, T.; Julian, R. R. J Am Chem Soc. 2011, 133, 6997. Viglino, E.; Lai, C. K.; Mu, X.; Chu, I. K.; Tureček, F. J. Am. Soc. Mass Spectrom.2016, 27, 1454. Syka, J.E.P.; Coon, J. J.; Schroeder, M. J.; Shabanowitz, J.; Hunt, D. F. Proc. Natl. Acad. Sci. U.S.A.2004, 101, 9528. Demirev, P. A. Rapid Commun. Mass Spectrom.2000, 14, 777. Zubarev, R. A.; Haselmann, K. F.; Budnik, B. A.; Kjeldsen, K.; Jensen, F. Eur. J. Mass Spectrom.2002, 8, 337. Zubarev, R. A.; Kruger, N. A.; Fridriksson, E. K.; Lewis, M. A.; Horn, D. M.; Carpenter, B. K.; McLafferty, F. W.J. Am. Chem. Soc.1999, 121, 2857.
(18) (19)
Papanastasiou, D.; Kounadis, D.; Lekkas, A.; Bozatzidis, A.; Orfanopoulos, I.; Raptakis, E.; Reinhardt-Szyba, M.; Damoc, E.; Makarov, A.; Zubarev, R. A. The Omni-trap: A new processing cell equipped with an extensive arsenal of ion activation techniques for top-down mass spectrometry, Presented at the ASMS conference on Mass Spectrometry and Allied Topics, Indianapolis, IN, June 4-8, 2017. Savitski M. M.; Kjeldsen, F.; Nielsen, M. L.; Zubarev, R. A. J. Am. Soc. Mass Spectrom. 2007, 18, 113. Nielsen, M. L.; Budnik, B. A.; Haselmann, K. F.; Olsen, J. V.; Zubarev, R. A. Chem. Phys. Lett.2000, 330, 558. Takahashi H, Sekiya S, Nishikaze T, Kodera K, Iwamoto S, Wada K, Tanaka K, Anal. Chem. 2016, 88, 3810. Shimabukuro, Y.; Takahashi, H.; Iwamoto, S.; Tanaka, K.; Wada, M. Development of a Compact Microwave Driven ICP Atom Source for Hydrogen Attachment/Abstraction Dissociation (HAD) of Tandem Mass Spectrometry, Presented at the ASMS conference on Mass Spectrometry and Allied Topics, Indianapolis, IN, June 4-8, 2017, WP352. Also, see WP 210 and WP 211. Zubarev, R. A.; Haselmann, K. F.; Budnik, B. A.; Kjeldsen, K.; Jensen, F. Eur. J. Mass Spectrom. 2002, 8, 337. He, M.; McLuckey, S. A. J. Am. Chem. Soc. 2003, 125, 7756. Kjeldsen, F.; Silivra, O. A.; Zubarev, R. A. Chem. Eur. J. 2006, 12, 7720. Van der Spoel, D.; Patriksson, A.; Adams, C. M.; Kjeldsen, F. Zubarev, R. A. J. Phys. Chem. B. 2007, 111, 13147. Marchese, R.; Grandori, R.; Carloni, P.; Raugei , S. PLoS Comput. Biol. 2010, 6, e1000775.
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