a2 Ion Derived from Triglycine: An N1-Protonated 4-Imidazolidinone

pubs.acs.org/JPCL

a2 Ion Derived from Triglycine: An N1-Protonated 4-Imidazolidinone Udo H. Verkerk,† Chi-Kit Siu,‡ Jeffrey D. Steill,# Houssain El Aribi,§ Junfang Zhao,† Christopher F. Rodriquez,†,^ Jos Oomens,#,r Alan C. Hopkinson,† and K. W. Michael Siu*† †

Department of Chemistry and Centre for Research in Mass Spectrometry, York University, 4700 Keele Street, Toronto, Ontario, Canada M3J 1P3, ‡Department of Biology and Chemistry, City University of Hong Kong, 83 Tat Chee Avenue, Kowloon Tong, Hong Kong, China, #FOM Institute for Plasma Physics, 3430 BE Nieuwegein, The Netherlands, §Applied Biosystems Deutschland GmbH, Frankfurter Strasse 129 B, 64293 Darmstadt, Germany, and rUniversity of Amsterdam, Nieuwe Achtergracht 166, 1018WV Amsterdam, The Netherlands

ABSTRACT Fragmentation of protonated peptides in the gas phase constitutes the basis for gas-phase sequencing of peptides using tandem mass spectrometry. Several mechanistic studies have indicated possible loss of bn ion sequence information as a consequence of macrocycle formation from internal nucleophilic attacks. Here, we show by infrared multiple-photon dissociation spectroscopy and density functional theory that the prototypical a2 ion generated from protonated triglycine is predominantly a cyclic N1-protonated 4-imidazolidinone. Cyclization resulting from internal nucleophilic attacks therefore may be a more general phenomenon than anticipated. SECTION Biophysical Chemistry

F

a protonated macrocyclic peptide; subsequent ring- opening of the macrocyclic ion can then occur at different residues, leading to the loss of sequence-specific information.7-10,13 Very recently, IRMPD has shown the b5 ion from the hexapeptide G5R (G = glycine and R = arginine) to have a macrocyclic structure.11 The classical fragmentation pathway for a bn ion is the elimination of CO from the protonated oxazolone, giving an ion that has been assumed to be a linear iminium ion.32-34 However, the a2 iminium ion, L1, has been predicted to easily cyclize to an N1-protonated 4-imidazolidinone, C1, via nucleophilic attack by the amino nitrogen on the iminium carbon (see Chart 1).32,33 This prediction predates the now much debated macrocyclic ion formation,4-13 and both cyclizations are initiated by nucleophilic attack by the terminal amino group.35,36 In this context, it is noteworthy that the a4 ion of leucine enkephalin is thought to exist as a mixture of structures, with linear ones being dominant and a cyclic structure being present as a minor component.37,38 Here, we present the first direct evidence provided by IRMPD spectroscopy, with support from MS/MS and a survey of the potential energy surface (PES) using density functional theory (DFT), that the a2 ion of protonated triglycine, the prototypical a2 ion, is predominantly cyclic and is an N1-protonated imidazolidinone, C1. Seven isomers were located at minima on the C3N2OH7þ PES using DFT calculations at the B3LYP/6-311þþG(d,p) level

ragmentation of protonated peptides in the gas phase constitutes the basis for sequencing peptides using tandem mass spectrometry (MS/MS).1-3 The mechanistic details of such reactions are a topic currently under intense scrutiny. New insights have been gained from infrared multiple-photon dissociation (IRMPD) spectroscopy and isotope-labeling studies that indicate possible loss of sequence information as a consequence of macrocyclic ion formation.4-13 Under low-energy collision conditions, dissociation of a protonated peptide is typically preceded by migration of a “mobile proton” between various basic sites, generating an ensemble of potential precursor ions.14,15 Subsequent chargeinduced cleavages of amide bonds yield a series of yn, bn, and an ions, which are used in peptide and protein identifications; these ions are compared with predicted fragment ions from peptides after in silico trypsinization of proteins whose sequences are available in a standard protein database. As yn, bn, and an ions play such important roles in protein identification, knowing the structures of these ions is important not only because of fundamental interest but also for a better understanding of fragmentation chemistry as it impacts upon sequence determination. While yn ions have been well recognized as protonated truncated peptides, there are competing structures for bn ions.16-22 Harrison et al. established that many b2 ions are protonated oxazolones,23-25 a result confirmed by IRMPD spectroscopy.26-28 Other b2 ions have the lower-energy protonated diketopiperazine structure.29-31 In recent observations,4-13 nucleophilic attack by the terminal amino group of the bn ion on the carbonyl carbon of the oxazolone has been shown to result in the formation of

r 2010 American Chemical Society

Received Date: December 28, 2009 Accepted Date: February 2, 2010 Published on Web Date: February 12, 2010

868

DOI: 10.1021/jz900464a |J. Phys. Chem. Lett. 2010, 1, 868–872

pubs.acs.org/JPCL

Scheme 1. Isomerization and Dissociation of the a2 Ion Derived from Protonated Triglycinea

Figure 1. IRMPD spectrum of the a2 ion derived from GGG (black trace) and IR absorption spectra for possible candidates calculated at the B3LYP/6-311þþG(d,p) level (red traces). The optimized theoretical IR spectrum is a linear combination of the IR spectra of the seven isomers (LCIR, blue trace). The individual isomer contributions to the LCIR are supplied under the structures, together with the relative enthalpies (ΔH0) and free energies (ΔG298, in parentheses) in kcal mol-1. Chart 1. Linear (L1) and Cyclic (C1) a2 Ion of Protonated Triglycine, GGG

(see Figure 1 and Scheme 1). The cyclic structure C1 was lower in enthalpy than the classical linear protonated iminium ion structure L1 by 9.6 kcal mol-1, with the barrier to interconversion only 8.9 kcal mol-1 above L1. The experimental IRMPD spectrum and the predicted IR absorption spectra for the seven isomers are also given in Figure 1. A visual inspection reveals that the spectrum computed for C1 most closely fits the experimental spectrum. The observed strong IR band at 1828 cm-1 is assigned to the CdO stretch of C1, predicted to be at 1825 cm-1. Stretching of the CdO bond produces a large change in dipole moment, resulting in this band being very intense (461 km mol-1). In addition, the predicted IR spectrum of C1 matches closely the IRMPD spectrum over the frequency range of 600-1700 cm-1. These spectral features strongly support the N1-protonated 4-imidazolidinone being the dominant isomer. The absence of IR bands with almost equal intensities at 1646, 1421, 1285, and 1146 cm-1 (the iminium group) and at 872 cm-1 (the amino group) eliminates L1 as a major contributor. Another linear iminium structure, L2, is 7.6 kcal mol-1 lower in enthalpy than C1; however, L2 is predicted to have an intense CdO stretching band at 1740 cm-1 (345 km mol-1) and a H-NþdCH2 bending band at


a Relative enthalpies (ΔH0) and free energies (ΔG298, in parentheses) are given in kcal mol-1.

1320 cm-1 (230 km mol-1). In the IRMPD spectrum, there is a low-intensity shoulder at around 1727 cm-1, possibly indicating the presence of L2 as a minor component. The N3protonated 4-imidazolidinone C2, the cyclic form of L2, can largely be excluded as a major contributor based on the observed weak absorptions at 677 and 1940 cm-1. Similarly, the absence of bands at 1699, 1486, and 1166 cm-1 excludes the O-protonated 4-imidazolidinone, C3. A theoretical spectrum expanded as a linear combination of the IR spectra of the seven isomers (LCIR) was obtained, using a program that incrementally changed the relative abundance of each of the seven isomer contributions. The calculated spectrum with the lowest standard deviation from

869


pubs.acs.org/JPCL

pneumatically assisted electrospray with N2 as the nebulizer gas. IRMPD spectroscopy was performed at the FOM-Institute for Plasma Physics “Rijnhuizen” in Nieuwegein, The Netherlands, using the Free Electron Laser for Infrared eXperiments (FELIX) facility.40 A laboratory-built Fourier transform ion cyclotron resonance mass spectrometer equipped with a Zspray (Micromass U.K. Ltd.) electrospray ionization source and a hexapole ion trap and guide was used to generate the a2 ion of protonated triglycine by adjusting the voltage difference between the sampling skimmer and the first ion guide. The a2 ion was isolated using a stored waveform inverse Fouriertransform (SWIFT) pulse and exposed to the IR laser beam. The spectrum shown in Figure 1 has been linearly powercorrected for diminished laser intensity in the high wavenumber range. Geometry optimizations and harmonic vibrational frequencies were calculated using the Gaussian 03 suite of programs at the B3LYP/6-311þþG(d,p) level of theory.41 All structures were characterized by harmonic frequency calculations, and intrinsic reaction coordinate calculations were used to determine the two minima associated with each transition structure. Frequency correction was applied using a scaling factor of 0.976, consistent with the scaling factors found appropriate in other studies of comparable systems.4,11,26-28,37 Band frequencies were convoluted using Gaussian profiles with a full width at half-maximum of 50 cm-1.

Figure 2. CID spectrum of the a2 ion derived from protonated GGG recorded at a center-of-mass collision energy of 3 eV.

the IRMPD spectrum was selected as the optimum fit. According to this procedure, the predominant isomer in the combination was C1 with a relative population of 0.55; the rest of the isomers were all minor components with relative populations of e0.2; see Figure 1. The CID spectrum of the a2 ion derived from protonated triglycine has only two discernible products at m/z 59 and 30 (Figure 2). The ion at m/z 59 has previously been shown to be a proton-bound dimer of methanimine (structure I in Scheme 1), and that at m/z 30 is protonated methanimine, the a1 ion, or the internal iminium ion (structure II).33,35,39 An examination of the PES (Scheme 1) shows that C1 can interconvert easily to L1 against a barrier of 18.5 kcal mol-1. The latter then dissociates by losing CO to give the protonbound dimer I at m/z 59 (barrier of 33.1 kcal mol-1 relative to C1). Dissociation of I is facile as protonated methanimine II is only 26.2 kcal mol-1 above I, and the barrier is the endothermicity of the reaction. Relative to C1, L2 is the second most abundant isomer at a relative population of 0.2. It is apparent from Scheme 1 that the most viable pathway of forming L2 from C1 is through C2 (as opposed to C3). The critical step is the interconversion between C1 and C2, against a barrier of 42.6 kcal mol-1; by comparison, the barrier between C2 and L2 is much lower at 18.6 kcal mol-1. It is, therefore, not surprising that C2 is present at a relative population of 0.05. Once formed, L2 is expected to be relatively stable as it is the isomer that has the lowest energy. L2 can interconvert to L3, L4, and C2 all with barriers e 26.2 kcal mol-1 (Scheme 1), in accordance with the IRMPD results of relative populations of 0.05 (Figure 1). Any possible dissociation products from these very minor isomers are not expected to be apparent in the fragmentation of a2, which is consistent with the experimental evidence in Figure 2. Details of the interconversion and fragmentation reactions are available in the Supporting Information.

SUPPORTING INFORMATION AVAILABLE Fragmentation of the a2 ion derived from protonated triglycine; proton migrations in protonated 4-imidazolidinone; and the isomerization between an O-protonated 4-imidazolidinone (C3) and a linear structure (L2) of the a2 ion. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author: *To whom correspondence should be addressed. Tel: (416)6508021. Fax: (416)736-5936. E-mail: [email protected].

Notes ^

ACKNOWLEDGMENT This study was supported by the Natural Sciences and Engineering Research Council of Canada and made possible by the facilities of the Shared Hierarchical Academic Research Computing Network. The skillful assistance of the FELIX staff is gratefully acknowledged.

REFERENCES (1)

TECHNICAL DETAILS Experiments were conducted on a PE SCIEX API III tandem mass spectrometer (Concord, Ontario, Canada). The a2 ion was generated via CID of the b2 ion that in turn was formed from protonated triglycine through in-source fragmentation. A 1 mM analyte solution in 70/30 water/ methanol with 1% acetic acid was continuously infused at a flow rate of 2 μL min-1 and ionized by means of


Deceased.

(2) (3)

870

Mann, M.; Hendrickson, R. C.; Pandey, A. Analysis of Proteins and Proteomes by Mass Spectrometry. Annu. Rev. Biochem. 2001, 70, 437–473. Aebersold, R.; Goodlett, D. R. Mass Spectrometry in Proteomics. Chem. Rev. 2001, 101, 269–295. Savitski, M. M.; Kjeldsen, F.; Nielsen, M. L.; Garbuzynskiy, S. O.; Galzitskaya, O. V.; Surin, A. K.; Zubarev, R. A. Backbone Carbonyl Group Basicities Are Related to Gas-Phase Fragmentation of Peptides and Protein Folding. Angew. Chem., Int. Ed. 2007, 46, 1481–1484.


pubs.acs.org/JPCL

(4)

(5)

(6)

(7)

(8)

(9)

(10)

(11)

(12)

(13)

(14)

(15)

(16)

(17) (18)

(19)

(20)

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. 2005, 127, 17154–17155. Harrison, A. G.; Young, A. B.; Bleiholder, C.; Suhai, S.; Paizs, B. Scrambling of Sequence Information in Collision-Induced Dissociation of Peptides. J. Am. Chem. Soc. 2006, 128, 10364–10365. Bleiholder, C.; Osburn, S.; Williams, T. D.; Suhai, S.; Van Stipdonk, M.; Harrison, A. G.; Paizs, B. Sequence-Scrambling Fragmentation Pathways of Protonated Peptides. J. Am. Chem. Soc. 2008, 130, 17774–17789. Vachet, R. W.; Bishop, B. M.; Erickson, B. W.; Glish, G. L. Novel Peptide Dissociation: Gas-Phase Intramolecular Rearrangement of Internal Amino Acid Residues. J. Am. Chem. Soc. 1997, 119, 5481–5488. Yague, J.; Paradela, A.; Ramos, M.; Ogueta, S.; Marina, A.; Barabona, F.; de Castro, J. A.; Vazquez, J. Peptide Rearrangement During Ion Trap Fragmentation: Added Complexity to MS/MS Spectra. Anal. Chem. 2003, 75, 1524–1535. Jia, C.; Qi, W.; He, Z. Cyclization Reactions of Peptide Fragment Ions During Multistage Collisionally Activated Decomposition: An Inducement to Lose Internal Amino Acid Residues. J. Am. Soc. Mass Spectrom. 2007, 18, 663–678. Mouls, L.; Aubagnac, J. L.; Martinez, J.; Enjalbal, C. Low Energy Peptide Fragmentations in an ESI-Q-TOF Type Mass Spectrometer. J. Proteome Res. 2007, 6, 1378–1391. Erlekham, U.; Bythell, B. J.; Scuderi, D.; Van Stipdonk, M.; Paizs, B.; Maitre, P. J. Infrared Spectroscopy of Fragments of Protonated Peptides: Direct Evidence for Macrocyclic Structures of b5 Ions. J. Am. Chem. Soc. 2009, 131, 11503–11508. Harrison, A. G. Peptide Sequence Scrambling Through Cyclization of b5 Ions. J. Am. Soc. Mass Spectrom. 2008, 19, 1776– 1780. Molesworth, S.; Osburn, S.; Van Stipdonk, M. Influence of Size on Apparent Scrambling of Sequence During CID of b-Type Ions. J. Am. Soc. Mass Spectrom. 2009, 20, 2174– 2181. Jones, J. L.; Dongre, A. R.; Somogyi, A.; Wysocki, V. H. Sequence Dependence of Peptide Fragmentation Efficiency Curves Determined by Electrospray Ionization/SurfaceInduced Dissociation Mass Spectrometry. J. Am. Chem. Soc. 1994, 116, 8368–8369. Dongre, A. R.; Jones, J. L.; Somogyi, A.; 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. 1996, 118, 8365–8374. Cordero, M. M.; Houser, J. J.; Wesdemiotis, C. Neutral Products Formed during Backbone Fragmentations of Protonated Peptides in Tandem Mass Spectrometry. Anal. Chem. 1993, 65, 1594–1601. Yalcin, T.; Harrison, A. G. Ion Chemistry of Protonated Lysine Derivatives. J. Mass Spectrom. 1996, 31, 1237–1243. Tsaprailis, G.; Nair, H.; Somogyi, A.; Wysocki, V. H.; Zhong, W. Q.; Futrell, J. H.; Summerfield, S. G.; Gaskell, S. J. Influence of Secondary Structure on the Fragmentation of Protonated Peptides. J. Am. Chem. Soc. 1999, 121, 5142–5154. Wysocki, V. H.; Tsaprailis, G.; Smith, L. L.; Breci, L. A. Mobile and Localized Protons: A Framework for Understanding Peptide Dissociation. J. Mass Spectrom. 2000, 35, 1399– 1406. Jonsson, A. P.; Bergman, T.; J€ ornvall, H.; Griffiths, W. J. Gln-Gly Cleavage: Correlation between Collision-Induced Dissociation


(21)

(22) (23)

(24)

(25)

(26)

(27)

(28)

(29)

(30)

(31)

(32)

(33)

(34)

(35)

(36)

871

and Biological Degradation. J. Am. Soc. Mass Spectrom. 2001, 12, 337–342. Farrugia, J. M.; O'Hair, R. A. J.; Reid, G. E. Do All b2 Ions Have Oxazolone Structures?: Multistage Mass Spectrometry and Ab Initio Studies on Protonated N-Acyl Amino Acid Methyl Ester Model Systems. Int. J. Mass Spectrom. 2001, 210, 71–87. Harrison, A. G. To b or Not to b: The Ongoing Saga of Peptide b Ions. Mass Spectrom. Rev. 2009, 28, 640–654. Yalcin, T.; Khouw, C.; Csizmadia, I. G.; Peterson, M. R.; Harrison, A. G. Why Are b Ions Stable Species in Peptide Mass Spectra?. J. Am. Soc. Mass Spectrom. 1995, 6, 1165– 1174. Yalcin, T.; Csizmadia, I. G.; Peterson, M. R.; Harrison, A. G. The Structures and Fragmentation of bn (n g 3) Ions in Peptide Mass Spectra. J. Am. Soc. Mass Spectrom. 1996, 7, 233–242. Nold, M. J.; Wesdemiotis, C.; Yalcin, T.; Harrison, A. G. Amide Bond Dissociation in Protonated Peptides: Structures of the N-Terminal Ionic and Neutral Fragments. Int. J. Mass Spectrom. Ion Processes 1997, 164, 137–153. Yoon, S. H.; Chamot-Rooke, J.; Perkins, B. R.; Hilderbrand, A. E.; Poutsma, J. C.; Wysocki, V. H. IRMPD Spectroscopy Shows That AGG Forms an Oxazolone b2þ Ion. J. Am. Chem. Soc. 2008, 130, 17644–17645. Oomens, J.; Young, S.; Molesworth, S.; Van Stipdonk, M. Spectroscopic Evidence for an Oxazolone Structure of the b2 Fragment Ion from Protonated Tri-Alanine. J. Am. Soc. Mass Spectrom. 2009, 20, 334–339. Bythell, B. J.; Erlekam, U.; Paizs, B.; Maitre, P. Infrared Spectroscopy of Fragments from Doubly Protonated Tryptic Peptides. ChemPhysChem 2009, 10, 883–885. Rodriquez, C. F.; Shoeib, T.; Chu, I. K.; Siu, K. W.; Hopkinson, A. C. Comparison Between Protonation, Lithiation and Argentination of 5-Oxazolones: A Study of a Key Intermediate in Gas-Phase Peptide Sequencing. J. Phys. Chem. A 2000, 104, 5335–5342. Perkins, B. R.; Chamot-Rooke, J.; Yoon, S. H.; Gucinski, A. C.; Somogyi, A.; Wysocki, V. H. Evidence of Diketopiperazine and Oxazolone Structures for HA b2þ Ion. J. Am. Chem. Soc. 2009, 131, 17528–17529. Savitski, M. M.; Falth, M.; Fung, Y. M. E.; Adams, C. M.; Zubarev, R. A. Bifurcating Fragmentation Behavior of Gas-Phase Tryptic Peptide Dications in Collisional Activation. J. Am. Soc. Mass Spectrom. 2008, 19, 1755–1763. Paizs, B.; Szl avik, Z.; Lendvay, G.; V ekey, K.; Suhai, S. Formation of a2þ Ions of Protonated Peptides. An Ab Initio Study. Rapid Commun. Mass Spectrom. 2000, 14, 746–755. Rodriquez, C. F.; Cunje, A.; Shoeib, T.; Chu, I. K.; Hopkinson, A. C.; Siu, K. W. M. Proton Migration and Tautomerism in Protonated Triglycine. J. Am. Chem. Soc. 2001, 123, 3006– 3012. Paizs, B.; Suhai, S. Combined Quantum Chemical and RRKM Modeling of the Main Fragmentation Pathways of Protonated GGG. II. Formation of b2, y1, and y2 Ions. Rapid Commun. Mass Spectrom. 2002, 16, 375–389. El Aribi, H.; Rodriquez, C. F.; Almeida, D. R. P.; Ling, Y.; Mak, W. W. N.; Hopkinson, A. C.; Siu, K. W. M. Elucidation of Fragmentation Mechanisms of Protonated Peptide Ions and Their Products: A Case Study on Glycylglycylglycine Using Density Functional Theory and Threshold Collision-Induced Dissociation. J. Am. Chem. Soc. 2003, 125, 9229–9236. El Aribi, H.; Orlova, G.; Rodriquez, C. F.; Almeida, D. R. P.; Hopkinson, A. C.; Siu, K. W. M. Fragmentation Mechanisms


pubs.acs.org/JPCL

(37)

(38)

(39)

(40)

(41)

of Product Ions from Protonated Tripeptides. J. Phys. Chem. B 2004, 108, 18743–18749. Polfer, N. C.; Oomens, J.; Suhai, S.; Paizs, B. Infrared Spectroscopy and Theoretical Studies on Gas-Phase Protonated Leu-enkephalin and Its Fragments: Direct Experimental Evidence for the Mobile Proton. J. Am. Chem. Soc. 2007, 129, 5887–5897. Polfer, N. C.; Bohrer, B. C.; Plasencia, M. P.; Paizs, B.; Clemmer, D. E. On the Dynamics of Fragment Isomerization in Collision-Induced Dissociation of Peptides. J. Phys. Chem. A 2008, 112, 1286–1293. Ambihapathy, K.; Yalcin, T.; Leung, H. -W.; Harrison, A. G. Pathways to Immonium Ions in the Fragmentation of Protonated Peptides. J. Mass Spectrom. 1997, 32, 209–215. For a review of the IRMPD spectroscopic technique and its applications, see: Polfer, N. C.; Oomens, J. Reaction Products in Mass Spectrometry Elucidated with Infrared Spectroscopy. Phys. Chem. Chem. Phys. 2007, 9, 3804–3817. Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Montgomery, Jr., J. A.; Vreven, T.; Kudin, K. N.; Burant, J. C.; et al. Gaussian 03, revision D.01; Gaussian, Inc.: Wallingford, CT, 2004.


872


a2 Ion Derived from Triglycine: An N1-Protonated 4-Imidazolidinone

Recommend Documents