Interconversion between 4-Imidazolone Ions; Isomers of [b4]+ Derived

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Interconversion between 4-Imidazolone Ions; Isomers of [b4]+ Derived from Protonated Tetraglycine K.H. Brian Lam,† Justin Kai-Chi Lau,†,‡ Cheuk-Kuen Lai,† Alan C. Hopkinson,† and K.W. Michael Siu*,†,‡ †

Department of Chemistry and Centre for Research in Mass Spectrometry, York University, 4700 Keele Street, Toronto, ON M3J 1P3, Canada ‡ Department of Chemistry and Biochemistry, University of Windsor, 401 Sunset Avenue, Windsor, ON N9B 3P4, Canada S Supporting Information *

ABSTRACT: Collision-induced dissociations of isotopically labeled protonated tetraglycines establish that the [b4]+ ion formed by loss of water from the second amide bond (structure II) rearranges to form N1-protonated 3,5-dihydro4H-imidazol-4-one (structure I), the product of water loss from the first amide bond. Structure II is slightly higher in energy than I (ΔH at 0 K is 5.1 kJ mol−1, as calculated at M06-2X/6-311++G-(d,p)), and the barrier to interconversion is 139.8 kJ mol−1 above I. The dominant dissociation pathway is the loss of methanimine (HN=CH2) from ion I with a barrier of 167.1 kJ mol−1, giving [GlyGlyGlyGly + H − H2O − HN=CH2]+, ion III; a minor channel, loss of NH3, has a slightly higher barrier (181.5 kJ mol−1). Using labeled glycine (13Cα) it was determined that loss of the imine is from the same residue as that from which water was initially lost. The collisioninduced dissociation spectra of ion III derived from both I and II were identical, and their energy-resolved curves were also very similar. Ion III fragments by losses of a glycine molecule (the dominant channel), a water molecule, and a glycine residue (57 Da), giving ions IV, V, and VII, respectively. Isotopic labeling established the origins of each of the neutral molecules that are lost. Using glycine (2,2 D2), rapid deuterium exchange was observed for both ions I and II for the α-hydrogens that are from the same residue as that from which the water had been eliminated.

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INTRODUCTION Collision-induced dissociation (CID) of protonated peptides produced by enzymatic digestion of proteins constitutes the basis for protein identification in proteomics.1−3 Protonated peptides are identified by the mass to charge ratios (m/z) of primarily the [bn]+ and [yn + 2H]+ ions4,5 that are formed by the cleavage of amide bonds. These dissociations are triggered by a cationizing proton that migrates between heteroatoms in the basic side chain and the peptide bonds, as described in the “mobile proton” model.6−11 For some protonated peptides, however, cleavages of peptide bonds are not the only major fragmentation pathways.12,13 Water loss is frequently observed and can be a significant pathway.13 Ballard and Gaskell proposed three possible sources for the loss of water: from a carboxylic acid group either at the C-terminus or an acidic side chain, as in aspartic or glutamic acid residues; from a hydroxyl group of a serine or threonine residue; or from an amide group (peptide bond) in the peptide backbone.13 In the fragmentations of protonated dipeptides and tripeptides that lack functional groups in the side chain that can lose water, dehydration appears to follow the first pathway and is predominantly from the C-terminal carboxylic acid group.14−18 The reaction is the © XXXX American Chemical Society

result of nucleophilic attack by the carbonyl oxygen of the preceding amide group on the carbon of the carboxylic acid and yields an oxazolone.14−17,19−24 For longer peptides, there can be substantial water loss from amide oxygens, and for the tetrapeptide [GlyGlyGlyGly + H]+ water is lost almost entirely from the amide backbone.25,26 Using 18O-labeled peptides, dehydration was found to be predominantly from the first amide oxygen, with water loss from the second amide oxygen being a minor channel.25,26 Infrared multiple photon dissociation spectroscopy and density functional theory (DFT) calculations showed the product ion to have an imidazolone structure (N1-protonated 3,5-dihydro-4Himidazol-4-one), structure I.25

Received: July 31, 2017 Revised: September 20, 2017 Published: September 25, 2017 A

DOI: 10.1021/acs.jpcb.7b07586 J. Phys. Chem. B XXXX, XXX, XXX−XXX

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The Journal of Physical Chemistry B

Figure 1. CID spectra of triglycine (a) after esterification at NCE = 19 and (b) with 18O-labeled at the first amide oxygen at NCE = 18. For brevity on the spectrum [GlyGlyGly-OMe + H]+ is abbreviated to [GGG-OMe + H]+. The precursor ion is denoted by an asterisk (*).

Figure 2. CID spectra of nominal [b4]+ ions formed by loss of oxygen from the (a) first amide and (b) second amide at NCE = 23. CID spectra in panels c and d are for the dissociation of m/z 200 ions at NCE = 26 derived from panels a and b, respectively. The precursor ion is denoted by an asterisk (*).

The operating pressure for the ion trap is approximately 2.05 × 10−5 Torr, resulting in multiple-collision conditions. Normalized collision energy (NCE, in %) is specified in the CID spectrum. The ESI probe was maintained at a voltage of 3.6 kV with the ion transfer tube operated at 225 °C. Nitrogen was used as the nebulizing gas at 3 arbitrary units and auxiliary gas at 1 arbitrary unit. Each solution was infused into the ESI source at a rate of 2 μL/min. Acetonitrile (Optima LC/MS grade) was purchased from Fisher Scientific (Ottawa, Canada) and was used without further purification. Milli-Q water (Type 1) was produced in-house using a Milli-Q Integral water purification system from Millipore (Billerica).

Herein, we report the collision-induced dissociations of the isomeric and nominal [b4]+ ions I and II, created by the loss of water from each of the first two amide groups in isotopically labeled [GlyGlyGlyGly + H]+. Our study led us to propose a hitherto unknown predissociation isomerization reaction between the two.

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EXPERIMENTAL SECTION Peptide Synthesis. All peptides were synthesized using solid-phase synthetic methods.27 H218O was purchased from Huayi Isotope Co. Fmoc-glycine Wang resin and Fmoc-glycine were purchased from Advanced ChemTech (Louisville, KY). Fmoc-glycine (2-13Cα) and Fmoc-glycine (2,2-D2) were purchased from Cambridge Isotope Laboratories (Xenia, OH). All other chemicals were purchased from Sigma-Aldrich (St. Louis, MO). 18O-labeled peptides were synthesized according to Marecek et al.28 Cleaved peptides were used without further purification. Mass Spectrometry. Samples were dissolved in 50/50 (v/v) acetonitrile/water, and experiments were performed on an Orbitrap Elite Hybrid Ion Trap-Orbitrap Mass Spectrometer from Thermo Fisher Scientific (Waltham), using an electrospray ionization (ESI) source. Helium was used as the collision gas for the ion trap while nitrogen was used for the C-trap.

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THEORETICAL METHODS

Geometry optimizations and calculations of harmonic vibrational frequencies were performed using the Gaussian 09 suite of programs (revision D.02)29 with the B3LYP functional based on Becke’s three-parameter exchange potential30,31 and the Lee, Yang, and Parr correlation functional.32 The standard Pople 6-311++G-(d,p) basis set was used for all calculations.33,34 All structures were characterized by harmonic frequency calculations; intrinsic reaction coordinate calculations were used to determine the two minima associated with each transitionstate structure.35 Subsequently all structures were optimized B

DOI: 10.1021/acs.jpcb.7b07586 J. Phys. Chem. B XXXX, XXX, XXX−XXX

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The Journal of Physical Chemistry B using the M06-2X functional36 and the same 6-311++G-(d,p) basis set. Throughout the text the relative energies quoted are enthalpies at 0 K (ΔH0° values) and free energies at 298 K (ΔG298°, typically given in parentheses). The overall conclusions from both levels of theory are very similar, and for simplicity, unless specified otherwise, only the M06-2X/6-311++G-(d,p) level calculations are used in the text.

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RESULTS AND DISCUSSION Loss of Water from [GlyGlyGly + H]+. The loss of water from [GlyGly + H]+ and [GlyGlyGly + H]+ is reportedly from the carboxylic acid group,14−17 while water loss from [GlyGlyGlyGly + H]+ is from an amide oxygen.25,26 This very different behavior prompted us to re-examine the fragmentation of [GlyGlyGly + H]+. First we found that fragmentation of the ester [GlyGlyGlyOMe + H]+ loses methanol and water, both in low abundance, with the second channel being the more minor (see Figure 1a). This encouraged us to synthesize Gly-(18O)GlyGly, where the N-terminal glycine was labeled with 18O, and in the dissociation of [Gly-(18O)-GlyGly + H]+ we observed losses of both H2O and H218O, again both in low abundance and with the latter being the lower of the two (Figure 1b). The barrier to loss of water from the carboxylic group from M06-2X/6-311++G-(d,p) calculations was found to be 128.2 kJ mol−1; this compares with an experimental value of 136 ± 9 kJ mol−1 and calculated values ranging from 107.3 to 136.2 kJ mol−1.15 The loss of water from the first peptide bond was higher in energy (166.7 kJ mol−1), and the transition state for the latter process was very similar to that for loss of water from [GlyGlyGlyGly + H]+ with the C-terminal glycine residue removed.26 Structures of the transition states for these two processes are given in Figure S1. Loss of Water from [GlyGlyGlyGly + H]+. Our current work confirmed that the water lost from [GlyGlyGlyGly + H]+ comes predominantly from the first amide oxygen and that losses from the second amide and the carboxylic acid group are both minor channels, as reported previously.25 We also labeled the third amide oxygen and found there is a negligible amount of water loss from this position (Figures S2 and S3). From DFT calculations, the imidazolidinone (structure I) formed by water loss from the first amide oxygen is lower in energy than that from the second amide (structure II) by 5.1 kJ mol−1, in accordance with the observation that removal of water from the first amide is preferred over that from the second. Interestingly, the CID spectra of the nominal [b4]+ ions created by removal of water from the first and second amide groups are essentially identical (Figure 2a,b). The most abundant product is the [b4 − HN=CH2]+ ion at m/z 200, with losses of NH3 and (CO + HN=CH2) being very minor channels. The CID spectra of the [b4 − HN=CH2]+ product ions (m/z 200) are also identical (Figure 2c,d), and the energy-resolved curves for the [b4 − HN=CH2]+ ions are also very similar (Figure S4). This suggests the two [b4]+ isomers rearrange to the same structure prior to fragmentation. Fragmentations and Structural Isomerization of [b4]+ Ions. Isotopically labeled glycine (13Cα) was used to identify which glycine residue is involved in the loss of methanimine. A comparison of Figures 2a and 3a shows that both the water and the methanimine came from the first glycine residue; similarly, a comparison of Figures 2b and 3b shows that both losses are from the second residue. From this we conclude that

Figure 3. CID spectra of [b4]+ ions formed by loss of oxygen from the (a) first amide and (b) second amide using 13C labeled tetraglycines at NCE = 23. The precursor ion is denoted by an asterisk (*).

Scheme 1. Mechanism for the Rearrangement of the [b4]+ Ions with Structures I and IIa

a

Enthalpies (at 0 K) and (free energies at 298 K), both in kilojoules per mole, as calculated at the M06-2X/6-311++G-(d,p) and B3LYP/6-311++G-(d,p) levels. All energies are relative to structure I.

the loss of the methanimine was always from the same residue as the one that had lost the initial water molecule. Loss of HN=CH2 from the imidazolone (structure I) formed by loss of water from the first amide is easily rationalized in terms of Cα−C bond cleavage adjacent to the fivemembered ring as it is analogous to the formation of an [a1]+ ion from a [b2]+ ion.37 Loss of HN=CH2 from the second residue requires rearrangement of structure II into I, thereby moving the second residue to the N-terminus. A possible pathway for rearrangement of II is given in Scheme 1. Proton transfer to the C

DOI: 10.1021/acs.jpcb.7b07586 J. Phys. Chem. B XXXX, XXX, XXX−XXX

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The Journal of Physical Chemistry B Scheme 2. Mechanisms for the Fragmentation and Isomerization of Structure Ia

a

Enthalpies (at 0 K) and (free energies at 298 K), both in kilojoules per mole, as calculated at the M06-2X/6311++G-(d,p) and B3LYP/ 6-311++G-(d,p) levels. All energies are relative to structure I. The first α-carbon is labeled by 13C for clarity.

nitrogen of the N-terminal amide is followed by nucleophilic attack by the imidazolone nitrogen on the amide carbon, resulting in cleavage of the amide bond and transfer of the N-terminal residue onto the imidazolone nitrogen. This second step has the highest barrier (139.8 kJ mol−1) on the overall profile from the M06-2X/6-311++G-(d,p) calculations. A subsequent attack on the newly formed imidazolone by the amino group of the NH2 of the first residue followed by ring opening leads to structure I with an overall exothermicity of 5.1 kJ mol−1. The pathways by which structure I dissociates or rearranges are given in Scheme 2. (i) The loss of HN=CH2 is the major pathway (pathway i) and occurs by direct cleavage of the exocyclic Cα−C bond to form ion III; the barrier to this pathway (167.1 kJ mol−1) is 27.3 kJ mol−1 above the barrier to isomerization to II. (ii) A 1,4-proton shift followed by nucleophilic attack by the carbonyl oxygen of the third amide group on the CH2 of the first residue in structure I displaces ammonia with a barrier of 181.5 kJ mol−1 (pathway ii). (iii) Direct loss of (CO + HN=CH2) from I could occur in two steps, first loss of CO (product not observed experimentally) followed by a higher energy step in which HN=CH2 is lost. The overall process has a much higher barrier (308.7 kJ mol−1, pathway iii) and clearly is not competitive with the loss of NH3. However, rearrangement of I into II moves the first residue into the second position (Scheme 1). This is followed by cleavage of the first amide bond of II which has a barrier of 191.5 kJ mol−1 (pathway iii′), and this provides a more competitive pathway for the combined loss of (CO + HN=CH2). Note that the neutrals lost in pathway iii′ do not contain labeled atoms and give the m/z 173 ion in Figure 3. Thus, the m/z 173

Figure 4. CID spectra of [b4 − HN=CH2]+ (structure III) containing 18 O and 13Cα labeling in the (a) first and (b) second glycine residue at NCE = 26. The precursor ion is denoted by an asterisk (*).

ion is not the product of a sequential loss from the ion at m/z 200. Fragmentations of Structure III at m/z 200. Isotopic labeling was used to study the dissociation of [GlyGlyGlyGly + H − H2O − HN=CH2]+, ion III. The spectra in Figures 4a,b are of ion III formed from I and II, respectively, where the glycine residue under consideration is labeled with 18O and 13 Cα. Scheme 3 shows possible fragmentation mechanisms of this ion. The product ions that retain the labeled atoms are formed by losses of glycine and water molecules, giving D

DOI: 10.1021/acs.jpcb.7b07586 J. Phys. Chem. B XXXX, XXX, XXX−XXX

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Scheme 3. Mechanisms for the Fragmentation of [GlyGlyGlyGly + H − H2O − HN=CH2]+ (Structure III) Illustrated Using the Products in Figure 4aa

a

Enthalpies (at 0 K) and (free energies at 298 K), both in kilojoules per mole, as calculated at the M06-2X/6-311++G-(d,p) and B3LYP/ 6-311++G-(d,p) levels. All energies are relative to structure III.

structures IV and V, respectively. The loss of glycine (pathway iv in Scheme 3) is facilitated by proton migration from the imidazolone ring to the amide nitrogen via protonation of the carboxy group. The direct 1,6-proton transfer from the imidazolone is constrained by the inflexibility of the ring and has a much higher barrier (199.9 kJ mol−1). A nucleophilic attack by the carbonyl oxygen of the imidazolone on the carbon of the protonated amide then leads to cleavage of the amide bond and generates an ion−molecule complex (barrier = 173.5 kJ mol−1). The α-hydrogen in the imidazolone ring is acidic, and the 1,2-proton transfer is assisted by using the glycine molecule as a transporter to produce a highly conjugated system, structure IV. Proton migration from the imidazolone ring to the peptide backbone can also lead to the loss of a water molecule from the carboxylic group at the C-terminus to produce an oxazolone structure V (pathway v). Further loss of CO from the oxazolone ring produces an imine, structure VI. The labeled atoms are lost as (C18O + HN=13CH2 in Figure 4) to give an ion at m/z 143. This is facilitated by nucleophilic attack

Figure 5. CID spectra of [b4]+ ions at NCE = 23 after water loss from the (a) first and (b) second amide oxygens using 18O-labeling and glycine (2,2-D2) in the same residue. The precursor ion is denoted by an asterisk (*). E

DOI: 10.1021/acs.jpcb.7b07586 J. Phys. Chem. B XXXX, XXX, XXX−XXX

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The Journal of Physical Chemistry B Scheme 4. Mechanism for α-Hydrogen Atom Scrambling in Structure Ia

a

Enthalpies (at 0 K) and (free energies at 298 K), both in kilojoules per mole, as calculated at the M06-2X/6-311++G-(d,p) and B3LYP/ 6-311++G-(d,p) levels. All energies are relative to structure I.

Notes

by the amide nitrogen at the C-terminus on the carbocation in the imidazolone ring to generate a bicyclic structure. Proton migration to the nitrogen of the original imidazolone ring weakens the N−Cα bond within the ring, and cleavage of this bond followed by breaking an amide bond results in the loss of CO + HN=CH2 (a glycine residue) containing the two isotopically labeled atoms from the N-terminus giving structure VII. Intramolecular H/D Exchange. While searching for possible mechanisms for water loss using isotopically labeled glycine (2,2 D2), scrambling of the hydrogens at the α-carbon was observed. Figure 5 shows that the α-hydrogens from the same residue that lost the initial water molecule can move to other basic sites. The energy barrier for scrambling of the α-hydrogen in structure I is 110.2 kJ mol−1 (Scheme 4), lower than the barriers to conversion to II and to dissociation. The acidity of the α-CH2 is attributed to the proximity to the positive charge that is formally delocalized between the two nitrogen atoms in the imidazolone ring of structure I. The energy-resolved curves for the deuterated [b4]+ ion in Figure S5 show that loss of methanimine with one deuterium is the most dominant at all collision energies.

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC) and made possible by the facilities of the Shared Hierarchical Academic Research Computing Network (http://www.sharcnet.ca) and the High Performance Computing Virtual Laboratory (http://www.hpcvl.org).

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CONCLUSION The nominal [b4]+ ion formed by loss of water from the second amide oxygen of tetraglycine, protonated imidazolidinone ion II, rearranges to the lower-energy isomer I prior to dissociation. A consequence of this rearrangement is that the loss of methanimine from the [b4]+ ions derived from [GlyGlyGlyGly + H]+ is always from the same residue from which water has initially been lost. The fragmentation pattern of [GlyGlyGlyGly + H − H2O − HN=CH2]+, ion III, follows several pathways that are common in the fragmentation of protonated peptides. Finally, the hydrogen atoms on the α-carbon of the residue from which water is lost undergo extensive scrambling in the imidazolidinone structure at lower energies than those required for rearrangement or dissociation.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcb.7b07586. Optimized transition states for water loss from triglycine, CID spectra of labeled protonated tetraglycine, and energy-resolved curves (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Justin Kai-Chi Lau: 0000-0001-8327-0644 F

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