Assessment of Gas Phase Basicities of Protonated Peptides by the

David J. Butcher, Keiji G. Asano, Douglas E. Goeringer, and Scott A. McLuckey. The Journal of Physical Chemistry A 1999 103 (43), 8664-8671. Abstract ...
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J. Phys. Chem. 1995,99, 10046-10051

10046

Assessment of Gas Phase Basicities of Protonated Peptides by the Kinetic Method Igor A. Kaltashov, Daniele Fabris, and Catherine C. Fenselau" Department of Chemistry, University of Maryland Baltimore County, 5401 Wilkens Avenue, Baltimore, Maryland 21228 Received: November 2, 1994; In Final Form: April 4, 1995@

Gas phase basicities of protonated peptides can be obtained, based on values of apparent basicities determined by a modified kinetic method, and the reverse activation energy barrier due to Coulombic repulsion can be determined by ion kinetic energy release spectrometry. The gas phase basicity for protonated bradykinin has been determined to be 217.8 f 1.7 kcallmol. The value of the reverse activation energy barrier due to Coulombic repulsion has been determined by MIKES experiments to be 13.6 f 1.4 k c d m o l . The influence of the distance between basic residues in the peptide chain on Coulombic repulsion and gas phase basicity was also investigated in a series of protonated isomeric peptides.

Introduction

ln([MH+]/[BH+]) = 1n(k,/k2)= (GB(M) - GB(B))/RTe,

Thermodynamic characteristics of protonated peptides and proteins are of paramount importance in biochemistry, because they govern both structure and function. Solvent effects are also very significant in water-based biological systems and often obscure intrinsic properties of macromolecules. The gas phase offers an alternative environment in which to evaluate thermodynamic properties of biopolymers unaffected by solvation. Since peptides and proteins usually carry multiple charges in their native state, gas phase chemists are challenged to study the properties of charged peptides and proteins. The introduction of electrospray by Fenn and co-workers' has given researchers a powerful experimental tool to produce multiply charged biomolecules and study their behavior in the gas phase. In recent years, the thermochemistry of rather small biomolecules has been studied extensively by means of mass spectrometry. Among other techniques, a kinetic method2 had been found to be a valuable tool in this kind of study, due to its ability to characterize thermally labile, nonvolatile molecules, while consuming only small amounts of material. In this method, the process under study is the decomposition of a proton-bound dimer, formed by a compound of interest, M, and a reference base. B:

VMH+ +

The relative abundances of MH+ and BH+ ions in a decomposition spectrum of the dimer reflect the difference in proton affinities (PA) of M and B3: ln([MH+I/[BH+I) = ln(k,/k2) = ln(Q#MH+.. .B/Q#BH+. .M)

+

( P A W ) - PA(B))/RTe, (3) where Teff represents the effective temperature of the dimer. Q#MH+-B and @BH+-M represent the partition functions for activated complexes in reactions 1 and 2, respectively. Entropy and enthalpy (PA) terms in (3) can be combined to give the free energy term, gas phase basicity (GB). Expression 3 may be rewritten then simply as

(e#) @

Abstract published in Advance ACS Abstracts, June 1, 1995.

0022-365419512099-10046$09.00/0

(4) The kinetic method has been shown during the past decade to be very useful in studying numerous classes of compounds, e.g. amine^,^ carboxylic acids: amino acids,'-' nucleos i d e ~ , ' ~and . ' ~ small peptide^.'^^'^ Recently, several groups have started to examine whether the classical methods of determining thermodynamic characteristics of neutral molecules (such as the kinetic and the bracketing methods) could be extended to studies of charged specie^.'^-'^ Since both of these methods are based on studying the charge separation process, the results of such studies are affected by the Coulombic repulsion of the two particles with like charges. Bursey and Pedersen" calculated the values of the electrostatic barriers for the reaction + H ~ N C H Z C H ~ C H ~ N HNR3 ~ + H2NCH*CH*CH*NH3+ NHR3+ to be in the tens of kcal/mol (for R = H, CH3). They also suggested that the value of this barrier could be roughly estimated as q2/r, where q is the elemental charge and r is a maximum molecular dimension. This approach was used by Bohme et al. in bracketing the gas phase acidities of some fullerene dications C60(XH),2+.18.'9 This paper presents a modification of the kinetic method that allows it to be extended to measure the gas phase basicities of cationic species in conjunction with determinations of Coulombic repulsion. In this case the process under study is the decomposition of a multiply protonated dimer (doubly charged for present considerations):

+

+

kp'\

MH+

+

-

BH+

The potential energy diagram for such a system was considered in ref 20. We reproduce it here (Figure 1) with only one modification: multiple minima at the bottom of the potential well represent the probable existence of isomeric forms of protonbound dimers involving large multifunctional compounds. Previous applications of the kinetic method have assumed the absence of reverse activation energy barriers for reactions 1 and 2. However, when dissociation of a doubly charged dimer occurs, electrostatic repulsion between MH+ and BH+ imposes a reverse activation energy barrier for reaction 6. The unimolecular rate constants kl' and k2' will be affected, therefore, by 0 1995 American Chemical Society

J. Phys. Chem., Vol. 99, No. 24, 1995 10047

Gas Phase Basicities of Protonated Peptides MH++B+H+

MH++B+H* [‘HM H’ B]’

1

7

I

V

[*HM.-H+-BI Figure 1. Potential energy surface diagram for unimolecular decomposition of a doubly charged proton-bound dimer.

the barrier:

where I3 represents the reverse activation energy barrier due to electrostatic repulsion. Thus, knowing the relative abundances of MH22+and MH+ ions, one can find the apparent gas phase basicity of MH+, which is offset by the Coulombic repulsion term: GB,,,(MH+) = GB(MH+)

+6

(8)

Thorough theoretical consideration^'^ show that the Coulombic term I3 may account for nearly all of the kinetic energy release when two singly charged ions (MH+ and BH+) are formed. In fact, the effect of the release of kinetic energy upon the decomposition of doubly charged ions was experimentally observed and discussed more than 50 years ago.2’ The kinetic energy release can now be measured rather accurately by a variety of mass spectral techniques. The most prominent are “coincidence” methods, utilized on time-of-flight mass spectrometers,22 and MIKES methods, utilized on sector mass spectrometer^.^^ The first applications of MIKES to probing the charge separation energy were reported a quarter century ag0,24325 and it is still being widely used in numerous analytical, structural, and physicochemical ~ t u d i e s . In ~ ~the , ~present ~ study, we used MIKES to determine experimentally the value of the Coulombic term I3 in the decomposition of a doubly charged dimer (6).

Experimental Section Methods. All experiments were performed on a JEOL HX1 lO/HXllO (JEOL, Tokyo, Japan) four-sector mass spectrometer (EBEB geometry) equipped with an electrospray ion source (Analytica of Branford, Branford, CT). In the present work multiply charged dimers were generated by electrospray ionization. Solutions of peptide mixtures (in water/methanol/ acetic acid) were electrosprayed at 2 pL/min. Nitrogen was introduced into the ion source as a curtain gas. Selecting rather mild conditions of spray ionization allowed us to achieve heterodimer intensities as high as 20% of those of monomer ions. In the present work only singly and doubly charged heterodimers were under investigation, although for larger peptides the formation of heterodimers of charge state as high as +4 has been detected. A heterodimer of interest was selected by the first two sectors of the mass spectrometer and introduced into the collision cell. No collision gas was added to the cell to acquire the decomposition spectra of metastable ions, while He or Xe was used for CID experiments. Dimer decomposition spectra were acquired using either E scans (for MIKES experiments) or BE scans (for all other experiments). Normally 50 to 120 scans were acquired for each spectrum. All spectra

were recorded using JEOL MP7000 data system. Molecular dynamics studies and calculation of lowest potential energy configurations of peptides were performed using CHARMm22/ QUANTA4.0 macromolecular modeling program (Molecular Simulations, Inc., Waltham, MA) on a Silicon Graphics Personal Iris workstation (Silicon Graphics, Inc., Mountain View, CA). Materials. Leucine-enkephalin and bradykinin were purchased from Sigma Chemical Co. (St. Louis, MO). Sequence isomers of bradykinin (peptides R2, R4, R7, and R8) were synthesized in The Biopolymer Lab (University of Maryland at Baltimore). Structures of all the peptides were verified by CID MSMS. l11,3,3-Tetramethylguanidine, tributylamine, tripropylamine, N,N-diisopropylmethylamine, and di-sec-butylamine were purchased from Aldrich Chemical Co., Inc. (Milwaukee, WI). All chemicals were used without further purification. Gas phase basicity values of all these reference bases are taken from ref 28.

Results and Discussion Low-Energy CID and Decomposition of Metastable Dimers. The metastable decomposition spectrum of a doubly charged dimer, m/z = 809, formed by bradykinin (RPPGFSPFR) and leucine-enkephalin (YGGFL) contains only three product ions: singly and doubly protonated intact monomers (Figure 2a). The difference in intensities of singly protonated bradykinin and leucine-enkephalin is attributed to differences in the transmission efficiencies for these two ions. The apparent gas phase basicity of the protonated bradykinin, GB,,,(MH+), can be estimated from the spectrum (see Figure 2a) using the value of the gas phase basicity of leucine-enkephalin,GB(B) = 231.3 f 0.5 kcal/ mol, which has been measured by the standard kinetic method using five reference bases: 1,1,3,3-tetramethylguanidine(GB = 234.8 kcal/mol), tributylamine (GB = 228.2 kcal/mol), tripropylamine (GB = 226.8 kcal/mol), N,N-diisopropylmethylamine (GB = 227.5 kcal/mol), and di-sec-butylamine (GB = 223.6 kcal/mol). The effective temperature of metastable proton-bound dimers usually lies in the range 300-700 K.3,29 We can assume that the effective temperature of the dimer, Teff, is 500 K. Even though this assumption is a rough approximation, it should not change the value of GB,,,(MH+) by more than 1 kcal/mol since in this case the [MH22+]/[MH+]ratio is close to unity. Taking all of the above into consideration, we obtain the value for the apparent gas phase basicity of singly protonated bradykinin, GB,,,(MH+) = 231.4 & 1 kcaumol. Collisionally induced dissociation (CID) of the dimer at low energies showed that the intact monomers remained the major fragments, accompanied by a small amount of fragmentation of covalent bonds (see Figure 2b). Figure 3 represents the relative abundances of doubly and singly protonated monomers at various collisional energies. A sharp rise of the graph between 0 and 3.3 eV as well as the nearly constant [MH22f]/ [MH+] ratio at higher collisional energies indicates that the entropy term in (7) is far from being 0. Cheng and co-workers suggested that the deviation of an entropy term from 0 indicates a substantial difference in the entropies of protonation of the two species.29 In the case of a doubly charged dimer, this is an indication that the entropies of the first and the second protonations are different. It is very likely that this is due to the difference in gas phase conformations of the singly and doubly protonated bradykinin. The singly protonated peptides are believed to be “folded” to stabilize the only proton in the system,29while the conformation of the doubly charged peptides in the gas phase could be rather extended due to electrostatic repulsion (see also discussion of the results of the molecular modeling studies). In general, more highly charged proteins have been found to exist in an extended conformation, as opposed to the lower charged, folded form^.^^.^^

10048 J. Phys. Chem., Vol. 99, No. 24, 1995

Kaltashov et al. rx25

a

531

1061

rx50

rx50

b !

60

-

20

-

S31

e 1061

500

600

700

am

900

I 000

I I00

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mlz Figure 2. Decomposition spectra of doubly charged dimers formed by bradykinin and leucine-enkephalin: dissociation of a metastable dimer (a), and CID at 10 eV in the center-of-mass frame (b). Scaling factors are indicated on top of each spectra.

Evaluation of the Value of Electrostatic Repulsion 6. It has been shown in ref 17 that the upper limit for the Coulombic term 6 can be approximated by the value of the kinetic energy release T upon the dissociation of the metastable dimer, since all of the electrostatic repulsion energy should appear as kinetic energy of the products. The value of Tcan be directly measured in mass-analyzed ion kinetic energy (MIKE) experiments on the decomposition of metastable ions:23

T = (y2m12eV)/(16xm,m,)(6El~2

(9)

where V is acceleration voltage, E is the center of the peak of the precursor ion mix+, 6 E is the peak width for the fragment ion m2?+,and m3 = ml - m2.

MIKE experiments were performed in the present study by scanning the electric field of the third sector of the tandem mass spectrometer to analyze the products of unimolecular decomposition of the doubly charged dimer, mlz = 809, formed by bradykinin and leucine-enkephalin (see Figure 4a). The width of the peak of singly protonated bradykinin (mlz = 1061), corrected for the width of the parent ion peak, is 6 E = 24.4 f 1.0 amu, which corresponds to a kinetic energy release T+z-+I = 0.63 d= 0.06 eV. This value, however, may represent only the upper limit for the Coulombic term 6, since it also may include the contribution due to the release of bond strains." The amount of non-Coulombic energy, converted into kinetic energy upon decomposition of the dimer (6) can be estimated

J. Phys. Chem., Vol. 99, No. 24, 1995 10049

Gas Phase Basicities of Protonated Peptides

0

l

o



i



)

I

,

I

,

l

io

20 30 40 50 a0 70 a0 eo 100 Collisional energy (center of maso frame), eV

Figure 3. Relative abundance of singly and doubly protonated intact monomers in the decomposition spectra of the doubly protonated dimer versus collisional energy in the center-of-mass frame.

TABLE 1: Apparent Gas Phase Basicity, GB, JMH+) and Gas Phase Basicity, GB(MH+), of Protonated seeptides and the Coulombic Rewlsion. 6 _ _ _ _ _ ~ ~~

GBapp(MH+),

peptide

kcal/mol“

R2

R4

230.2 f 1 230.2 & 1

R7

230.0 i 1

R8

231.7 +z 1 231.4 f 1

R9

GB(MH+),

6, kcaVmol 13.4 i 1.4 12.7 & 1.4 13.1 i 1.4 12.9 i 1.4 13.6 1.4

kcaVmol 216.8 f 1.7 217.5 1.7 216.9 +z 1.7 218.8 f 1.7 217.8 f 1.7

Calculated assuming Teff = 500 K. from a peak width of doubly protonated bradykinin (m/z = 531), Le. as a kinetic energy release upon decomposition of the same dimer without charge separation. The width of this peak, corrected for the width of the parent ion peak, is 6E531 = 3.0 f 0.4 amu, which corresponds to a kinetic energy release T+2-+2= 0.04 & 0.01 eV. The charge repulsion term may be then estimated simply as 6 = T+2-+1 - T+2-+2 = 0.59 f 0.06 eV (or 13.6 f 1.4 kcal/mol). The experimental error in measuring 6 was determined as a deviation in a series of five experiments. The value of the gas phase basicity of singly protonated bradykinin can be found now according to (8): GB(MH+) = 217.8 & 1.7 kcal/mol. It is worth mentioning that the gas phase basicity of neutral bradykinin exceeds 245 kcal/mol, the highest value that can be measured using common organic base reference^.^^,^^

Effect of the Location of Basic Residues on the Gas Phase Basicity of a Charged Peptide. To test the notion that protons can be localized in multiply charged peptides and to study the influence of the distance between basic residues on the gas phase basicity of a peptide, the following sequence of peptides has WPRGFSPF (R4); WPGFSbeen used: W P G F S P F (U); IRPF (R7); RPPGFSPRF (R8); WPGFSPFR (R9, or native bradykinin). The only difference among these isomers is the position of the second Arg residue in the peptide chain. Arginine is the most basic amino acid, whose gas phase basicity exceeds that of its closest follower, histidine, by more than 10 kcal/moL8 Therefore, it is often assumed that Arg residues in peptides and proteins are the most likely sites of protonation. The reference compound (leucine-enkephalin) does not contain Arg residues. We estimated the values of apparent gas phase basicities GB,,,(RnH+) of singly protonated peptides in the series by monitoring the [RnH2*+]/[RnH+] ratio in the unimolecular decomposition spectra of metastable doubly charged dimers formed by each of the Rn peptides with leucine-enkephalin,

[Rn+B+2HI2+ (mlz = 809). The data showed a slight increase in the apparent gas phase basicity of RnH+ with increase of the distance between the Arg residues (see Table 1 ) . For all of the listed peptides MIKES experiments were performed, analogous to those described in the previous section. Figure 4 shows MIKE spectra of the decomposition products of metastable dimers formed by bradykinin (a) and R2 (b) with leucineenkephalin. Surprisingly enough, the Coulombic terms for all of these peptides, as well as for R9 (native bradykinin), were found to be similar within their experimental errors and to be independent of the distance between the two Arg residues (see Table 1). These experimental results were then compared to the results of molecular modeling studies. Calculations were carried using the CHARMm22 empirical force field33 for peptides R2 and R9. We assume that the gas phase conformations of these peptides in the transition state [ + H M O*H+*.B]*(the maximum on the right side of the potential energy diagram on Figure 1) closely approximate conformations of the isolated peptides. Our assumption is that in the transition state the only interaction between the peptide of interest MH22+ and the molecule of leucine-enkephalin B is through the “shared’ proton. This means that the peptide of interest is not “solvated” by the neighboring molecule, and so its conformation must be unaffected. Both peptides’ structures (R2 and R9) were built in QUANTA4.0 using the CHARMm22 library of standard Lamino acid residues. A proton was removed from the Nterminal and placed on the C-terminal, since there is evidence that peptides do not exist in the gas phase as zwitterion^.^^ For peptide R9, the two “extra” protons were located on the guanidine groups of the Arg residues, since these sites are the most basic and the most remote from each other among all functional groups of the peptide. For peptide R2, two models were studied. In the first one, both protons were located on the guanidine groups of the adjacent Arg residues. In the second model, one proton was placed on a guanidine group of the Argl residue, while the second one was attached to the carboxylic carbonyl oxygen atom of the Phe9 residue. We chose the carboxy group of Phe9 as a second protonation site because this functional group is the most remote from the N-terminal of the peptide and it is reported to be more basic than amide nitrogen.35 The total charges of the peptides were adjusted to 2.0 using carbons and nonpolar hydrogens to smooth the charge. These starting linear conformations were then subjected to molecular dynamics simulatiodenergy minimization using the following procedure. The starting conformation was energy minimized (method of steepest descents, 200 steps) to remove possible steric overlaps. The molecule was then heated from 0 to 750 K during 1.05 ps, equilibrated at that temperature for 1 ps, and followed by dynamics simulation for 1 ps. The time step for each of the dynamics procedures was ps. The final conformation was then again energy minimized (method of steepest descents, at least 5000 steps to root mean square gradients well below 0.1). The whole dynamics/minimization cycle was repeated at least five times for each peptide (until the difference in free energy for two consecutive cycles was less than 2 kcal/mol). The final conformations of the peptides are shown in Figure 5. The intercharge distance in R9 can be approximated as 26 A (Figure 5a), which is very close to the intercharge distance in R2 (24 A) if we choose Arg’ and Phe9 as the protonation sites (Figure 5b). Given these approximations for the intercharge distance in both peptides (R9 and R2), we can estimate the value of the electrostatic repulsion simply as 6caIc = q2/rmax(0.55 and 0.60 eV, respectively). These values are very close to experimentally found Coulombic repulsion terms for R9 and R2 (0.59 It 0.06 and 0.58 Z!C 0.06 eV,

Kaltashov et al.

10050 J. Phys. Chem., Vol. 99, No. 24, I995 rx50

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m/Z Figure 4. MIKE spectra of the decomposition of doubly charged metastable dimers formed by bradykinin (a) and peptide R2 (b) with leucineenkephalin. Scaling factors are indicated on top of each spectra.

respectively). If, however, the protons are placed on the Arg’ and Arg2 residues in R2 (Figure Sc), the intercharge distance becomes much less (14 A), and the corresponding repulsion energy (1.03 eV) becomes significantly larger than the experimentally found value. This suggests that in the transition state of the complex [R2+B+2HI2+ the protonation sites on R2 are Arg’ and Pheg, not A r g ’ and Arg2. Interestingly, the CHARMm

calculations also indicate that the protonation with maximum charge separation leads to an energetically more favorable (by almost 60 kcal/mol) conformation due to more effective “selfsolvation” of the Arg2 side chain by the peptide and reduction in the intercharge Coulombic repulsion. Experimentally determined values of apparent gas phase basicities and the Coulombic repulsion terms for protonated

Gas Phase Basicities of Protonated Peptides

J. Phys. Chem., Vol. 99, No. 24, 1995 10051 a -c-tmn

of molecular modeling studies of the doubly charged peptides, suggesting that the protons are not necessarily located on the most basic sites of peptides in proton bound dimers, if the alternative of a greater intercharge distance is available.

4

cPhe'

Ro'-

Acknowledgment. The authors are grateful to Dr. Zhuchun Wu and Dr. Michele Kelly for very helpful discussions. The work was supported by a grant from the National Science Foundation. References and Notes

u -m

Figure 5. Conformations of R9 (a) and R2 (b, c) doubly charged peptides obtained using the CHARMm22/QUANTA4.0macromolecular modeling program.

peptides R2, R4, R7, and R8 were used to determine their gas phase basicities (Table 1).

Conclusion

A kinetic method has been extended in combination with kinetic energy release studies to measure the thermodynamic parameters of protonated bradykinin, providing values for both the apparent gas phase basicity and the reverse activation energy barrier due to electrostatic repulsion. The apparent gas phase basicity of protonated bradykinin (RPPGFSPFR) is estimated at 231.4 f 1 kcal/mol. The Coulombic repulsion term, determined by MIKE experiments, was found to be 13.6 f 1.4 kcal/mol, giving the value of the gas phase basicity for singly protonated bradykinin, GB(MH+) = 217.8 k 1.7 kcal/mol. Decreasing the distance between the two Arg residues in the bradykinin chain leads to a slight decrease in the apparent gas phase basicities of the peptides, but no such trend is observed for the value of the Coulombic repulsion 6. Experimentally found intercharge repulsion is in good agreement with the results

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