Retention Prediction of Peptide Diastereomers in Reversed-Phase

Publication Date (Web): September 4, 2012. Copyright © 2012 American Chemical Society. *R. Chyu Ruaan: E-mail: [email protected]; fax, +886-3-280-4...
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Retention Prediction of Peptide Diastereomers in Reversed-Phase Liquid Chromatography Assisted by Molecular Dynamics Simulation C. Wei Tsai,† W. Yih Chen,*,† and R. Chyu Ruaan*,†,‡ †

Department of Chemical and Materials Engineering, National Central University, Jhong-Li, Taiwan 32001 R&D Center for Membrane Technology, Department of Chemical Engineering, Chung Yuan Christian University, Jhong-Li, Taiwan 32001

‡

S Supporting Information *

ABSTRACT: In this study, we explored the relationship between the retention factors and structural flexibilities of peptide diastereomers in reversed-phase chromatography (RPC) based on thermodynamic interpretations. The RPC retention order of antimicrobial peptides, IL-K7F89 (HILPWKWKFFPWRR-NH2), and its four diastereomers were well correlated with the order of their conformation energies in elution solvent. In particular, when the composition of the sample loading solvent was altered, the retention order changed accordingly. The thermodynamic analysis revealed that the peptide adsorption was driven by adsorption enthalpy, but the retention order was dominated by adsorption entropy. To further understand the relationships between the retention factor and conformation energy, the intramolecular van der Waals energy of peptides and the ordered water molecules associated with peptides were analyzed by all-atom molecular dynamics (MD) simulation. The results showed that the flexible peptide with larger conformation energy had weaker intramolecular hydrophobic interaction and associated with more ordered water molecules. For this peptide diastereomer set, the elution difference is derived by the difference in adsorption entropy gain from repelling the ordered water molecules around the peptide. Consequently, we suggested that the separation of peptide diastereomers could be achieved by RPC, and the elution order could be predicted by their structural flexibilities.

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INTRODUCTION Reversed-phase liquid chromatography (RPLC) is widely used for the analysis of various chemical substances.1−5 In order to speed up method development, it is important to predict the retention of the target product and possible contaminants. Most predictions rely on the understanding of the physicochemical properties of solutes and solvents. Several approaches have been investigated, such as Snyder’s wellknown P′ scale,6 solubility parameter developed by Hansen and Karger et al.,7−9 and the MOSCED scale from Eckert.10,11 However, most of the models are only valid for a certain subsets of chemicals. The complexity of molecular interaction leads to much more complicated models, such as the linear solvation energy relationship (LSER)12−15 and quantitative structure−retention relationships (QSRRs).16−18 Still, these approaches fail to predict the retention of peptides, especially of the sequence shuffled peptide sets. Hodges et al. categorized two types of sequence-dependent effects resulting in deviations from predictable RP-HPLC retention behavior of peptides, namely, conformation effects and nearest-neighbor effects.19 The conformational effects describe the deviation of peptide hydrophobicity from the predicted value because peptides adopt a unique conformation on interacting with the hydrophobic stationary phase. The © 2012 American Chemical Society

nearest-neighbor effects result in sequence-dependent variability of peptide retention but are independent of defined secondary structures such as α-helix or β-sheet. Hodges’ group has extensively studied the conformation effect of peptides adopting an α-helical structure.20−24 However, we still lack a general rule for predicting the sequence effect on peptide retention on RPLC. We previously proposed a simple strategy for predicting the retention order of sequence shuffled peptides.25 Peptides with a higher conformation energy in solution are considered to have higher retention factors in the reverse phase column. Two possible mechanisms contribute to the prolonged retention caused by high conformation energy. One is the adsorption enthalpy enhanced retention: peptides with higher conformation energy in solution have either lower deconformation energy or lower interaction enthalpy upon adsorption. Lower deconformation energy or lower interaction enthalpy results in lower overall adsorption enthalpy; consequently, this extends column retention. The other possible mechanism is that the adsorption entropy enhanced retention: peptides with higher Received: March 21, 2012 Revised: July 28, 2012 Published: September 4, 2012 13601

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Chem-Lab data station (SISC, Taiwan). The retention time of nonretained molecules in the RPC column was measured by injecting 0.1 mg/mL sodium nitrate. All measurements were performed at least three times, and the average values were reported. Molecular Modeling. All-atom MD simulation systems were constructed with an extended peptide solvated in either water or 30% ACN/water aqueous solution by the AMBER program.29 The Nterminus and lysine residues were protonized for modeling the peptides in elution solvent containing 0.1% TFA. The simulation system contains 4360 water molecules, and an additional 645 ACN molecules were used to mimic the 30% ACN/H2O aqueous solution. Before MD simulation, 2500 steps of energy minimization were performed to remove the bad contacts of the initial configurations. The simulation systems were pre-equilibrated after energy minimization by the modified AMBER 94 force field for 0.5 ns. For details of MD simulation, the potential functions of the peptide and ions were modeled using the AMBER 94 protein all-atom force field. The water and ACN molecules were treated with the TIP3 model30 and the six sites flexible molecular model,31 respectively. All-atom MD simulations were performed using the parallel MD NAMD 2.6 software32 with an NPT ensemble under three-dimensional periodic boundary conditions. The simulation pressure was controlled at 1 bar using the Langevin piston Nosé−Hoover method;33 the temperature was controlled at 298 K through Langevin dynamics. Cutoffs of 12 Å were applied to calculate the pairwise interactions and to generate the list of pairs. The nonbonded neighboring list was updated every 20 steps. Long-range electrostatic interaction was treated by the particle-mesh Ewald (PME) technique.34 The hydrogen atom-involved covalent bonds were constrained by the SHAKE algorithm,35 allowing an integration time step of 1 fs. The visualization of the atomic configurations and the analysis of interaction energy were determined by VMD software.36 The simulation time ran for 10 ns. Average energies were used from the last 0.5 ns of the MD simulation. To calculate the number of ordered water molecules close to a peptide, we evaluated the H-bond between water molecules near the peptide (the O atom of water within 8 Å from the peptide). The hydrogen bond formed between two water molecules was defined by the following: (1) the distance between donor (O) and receptor (H) was below 3.5 Å and (2) the hydrogen-donor−acceptor angle larger than 135°. The ordered water molecule was defined by the number of H-bonds per water. The water molecule that formed 3 H-bonds with its neighboring waters was defined as an ordered water. The region of 8 Å surrounding a peptide contained roughly two layers of water molecules, and the 3 H-bonds were defined by taking the value of the icelike water which normally forms 3.47 H-bonds.37

conformation energy in solution have more associated solvent. More associated solvent results in a higher number of repelled solvent after adsorption, which elevates adsorption entropy and thus enhances column retention.26,2726,27 The retention of two sets of sequence shuffled peptides in a C18 reverse phase column has been investigated.25 It has been found that peptides with higher conformation energy in solution exhibit higher retention factors. In this study, we extended this hypothesis to the column retention of peptide diastereomers. Four C-terminal amidated peptide diastereomers composed of 13 amino acids are designed from the potential antimicrobial IL-K7F89 peptide,28 which is an indolicidin derivative with lower hemolytic activity. These peptide diastereomers (sequence was shown in Table 1), which Table 1. Sequence of IL-K7F89 Peptide and Its Diastereomers peptide

sequencea

IL-K7F89 DL2 DW4 DK7 DW11

H-ILPWKWKFFPWRR-NH2 H-ILPWKWKFFPWRR-NH2 H-ILPWKWKFFPWRR-NH2 H-ILPWKWKFFPWRR-NH2 H-ILPWKWKFFPWRR-NH2

a

The one-letter amino acid core is used. The underlined residue represents the D-amino acid. The peptides used were amidated at the C-terminus.

have a single amino acid mutation from the native L- to Dform, possess the same amino acid sequence and subsequently the same residue-based hydrophobicity as the virgin IL-K7F89. However, the 3D structures of these peptide diastereomers may be different in their effect on the retention behaviors in RPC. Thus, the structures of these peptides in elution solvent were also measured and simulated by CD measurements and an allatom molecular simulation approach, respectively. Furthermore, the conformation energies of these peptides in solution are estimated by all-atom molecular simulation and are compared with their elution order in a C18 reverse phase column. The possible mechanism relating conformation energy to elution order was analyzed by adsorption enthalpy and entropy obtained from thermodynamic measurement and the number of ordered water molecules obtained from molecular modeling.

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RESULTS AND DISCUSSION Retention Behaviors of IL-K7F89 Peptide and Its Diastereomers in RP-HPLC. Four peptide diastereomers were designed by single residue substitution of D-form amino acids at the Leu2, Trp4, Lys7, or Trp11 position, and the names of these peptide diastereomers were designated as DL2, DW4, DK7, and DW11, respectively. Table 1 shows the sequence and mutation position of these peptides. The retention times of the IL-K7F89 peptide and its four diastereomers in a C18 reverse phase column were measured under an isocratic condition in chromatography operation. The retention factor (ln k′) could be calculated by eq 1:

MATERIALS AND METHODS

Chemical Components. IL-K7F89 peptide and its four diastereomers including DL2, DW4, DK7, and DW11 (sequence listed in Table 1) were purchased from MDBio, Inc. (Taipei, Taiwan) with the purities higher than 95%. The HPLC-grade acetonitrile (ACN) and trifluoroacetic acid (TFA) were purchased from J.T. Baker (USA) and Aldrich (Germany), respectively. Reversed-Phase Chromatographic Operation. RPC experiments were performed by a HPLC system consisting of two Lab Alliance series IV pumps (USA) and a UV detector (Lab Alliance model 520 UV, USA). The commercial Ace 5 C18-300 RPC column with the length of 150 mm and i.d. of 4.6 mm (Advanced Chromatography Technologies, Scotland) was used. The temperature of column was controlled by a water jacket surrounding the column. Each peptide with the final concentration of 0.1 mg/mL was dissolved in acetonitrile/water solutions (0 or 30 (v/v) %) containing of 0.1% TFA. A volume of 20 μL of the peptide sample was injected and eluted isocratically at 30% (v/v) acetonitrile. The retention of peptides was recorded at five column temperatures (20, 25, 30, 35, and 40 °C). The peptide signals were monitored at UV 215 nm and recorded through a

⎛ t − t0 ⎞ ln k′ = ln⎜ R ⎟ ⎝ t0 ⎠

(1)

where tR and t0 are the retention time of peptide and nonretained molecule in the RPC column, respectively. The RPC experiments were performed using the isocratic elution condition of 30% (v/v) ACN containing 0.1% (v/v) TFA at 25 °C. We also discussed the sample loading solvent on 13602

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times are listed in Table 2. The chromatograms revealed that DL2 diastereomer was eluted first and DK7 was slightly behind. The diastereomer DW4 appeared in the third place, followed by the virgin IL-K7F89. DW11 was found to be the most retained one. However, the residue-based hydrophobicity of these peptides could theoretically be the same since they had the same primary structure (amino acids sequence). We actually observed that the peptides exhibited different retention behaviors in the C18 reverse phase column. Apparently, the peptide retention was not completely controlled by the overall hydrophobicity. The secondary structure of peptides may have affected their retention in the RP column.38 We suspected that the D-form substitution altered the secondary structure of a peptide in solution and thereby altered the interaction between the peptide and hydrophobic resins. Thus, we further measured the secondary structure of IL-K7F89 and its diastereomers by a circular dichroism (CD) spectrometer. The CD spectra revealed that all the peptides in 30% (v/v) ACN solutions exhibited random coil structures (please see the Supporting Information), and the secondary structures of peptide diastereomers were slightly different. Thus, the results indicate that the conformations of peptide diastereomers may play a role in their retention in RPC. On the other hand, chromatograms of sample loading solvent by 0% (v/v) ACN and elution solvent by 30% (v/v) ACN, shown in Figure 1B, and their retention time, listed in Table 3,

the retention of peptide diastereomers; the peptide samples were prepared in either 0% or in 30% (v/v) ACN aqueous solution containing 0.1% (v/v) TFA. The chromatograms of peptides that were prepared at 30% (v/v) ACN were also eluted by the same solution shown in Figure 1A. The retention

Table 3. Retention Time (tR), VDW Energy, and Conformational Energy of Peptides in 0% ACN peptide

tR (min)a

EVDW (kcal/mol)b

Econf (kcal/mol)c

DW11 IL-K7F89 DW4 DL2 DK7

5.36 4.52 4.68 3.36 3.14

−3.48 −5.81 −4.45 −9.29 −10.75

117.21 109.00 109.92 92.63 86.17

a

tR is obtained from the RPC experimental measurements in 30% ACN, and the sample loading solvent is 0% ACN. bEVDW, van der Waals energy among the hydrophobic residues in peptides from MD simulation calculation; average energies were used from the last 0.5 ns of the MD simulation. cEconf, conformation energy of peptides in sample loading solvent from MD simulation calculation; average energies were used from the last 0.5 ns of the MD simulation.

Figure 1. Chromatograms of IL-K7F89 peptide and its diastereomers in RP-HPLC. The peptide was eluted by 30% ACN/H2O containing of 0.1% TFA under the flow rate of 1 mL/min at 25 °C. (A) The sample loading solvent is the same as the elution solvent. (B) The sample solvent is 100% H2O containing of 0.1% TFA.

provides evidence that differs from this assumption. Although the diastereomer DW11 was still the most retained one, the

Table 2. Retention Time (tR), Thermodynamic Parameters, VDW Energy, Conformational Energy, and Order Water Molecules of Peptides in 30% ACN peptide

tR (min)a

DW11 IL-K7F89 DW4 DK7 DL2

5.14 4.69 4.10 2.78 2.74

ΔH° (kJ/mol)a TΔS°* (kJ/mol)a ΔG°* (kJ/mol)a −8.583 −10.923 −10.725 −21.744 −24.234

−6.291 −8.806 −8.936 −20.837 −23.467

−2.292 −2.117 −1.790 −0.907 −0.767

EVDW (kcal/mol)b −3.63 −3.73 −4.12 −4.75 −5.01

Econf (kcal/mol)c total associated H2Od 112.73 104.22 101.38 93.86 87.10

405.66 378.24 364.95 333.85 319.66

ordered H2Oe 36.700 34.140 31.886 28.057 25.691

tR, ΔHo, TΔSo, and ΔGo are obtained from the RPC experimental measurements as the sample loading and elution solvents are 30% ACN. bEVDW, van der Waals energy among the hydrophobic residues in peptides. EVDW is calculated from MD simulation. cEconf, conformation energy of peptides in sample solvent. Econf is calculated from MD simulation. dAverage number of total associated water molecules around the peptide within 8 Å from the last 0.5 ns of the MD simulation. eAverage number of ordered water molecules around the peptide within 8 Å from the last 0.5 ns of the MD simulation; the average values were calculated by counting of the H-bond per water above 3 H-bonds. The hydrogen bond formation between each water molecule is based on the distance between the donor−acceptor that is below 3.5 Å, and the hydrogen-donor−acceptor angle is larger than 135°. a

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Figure 2. Snapshots of IL-K7F89 peptide and its diastereomers. The configurations of IL-K7F89 peptide and its diastereomers from MD simulation were shown in (A) 30% ACN/H2O and (B) 100% H2O. The peptide backbone was represented by a ribbon. Positively charged residue, hydrophobic residue, and the D-form replaced residue were colored in blue, gray, and yellow, respectively.

retention order altered. The diastereomer DK7 was eluted first this time and followed by DL2. The virgin IL-K7F89 peptide came in third place with DW4 following. These results indicated that the elution order could be altered by the composition of the sample loading solvent, and that it is important to examine why and how sample loading solvent affects the retention of peptide in RPC. Furthermore, the CD spectra revealed that all the peptides exhibited random coil structures, regardless of whether they were in 0 or 30% (v/v) ACN solutions (please see the Supporting Information). The results revealed that the secondary structures of peptide diastereomers were slightly different, but it was difficult to find any correlation between CD spectra and their retention order in the C18 column. Another approach for understanding the structural information on the retention order of peptide isomers is provided by molecular modeling. Conformation Energy Estimated by All-Atom Molecular Dynamics Simulation. In our previous study, we proposed that the retention order of sequence shuffled peptide isomers could be predicted by their conformation energy in elution solvent. The peptide with higher conformation energy in solution possessed a more flexible structure and was supposed to have a longer retention time in the C18 column.25 We, therefore, tried to test the hypothesis on peptide diastereomers differed by only one or two D- or L-amino acids. The conformation energy (Econf) of a peptide in solution consists of both nonbonded (Enonbonded) and bonded energies (Ebonded): Econf = Enonbonded + E bonded

the data was analyzed from the last 0.5 ns of the MD simulation. The steric configurations of IL-K7F89 and its diastereomers in solution are shown in Figure 2. The simulation results were in accordance to the CD spectra, and the peptides adopted random coil structure in solution. However, the snapshots showed that the structures of these peptides were all different. The conformation of each peptide in 30 v/v% ACN solution was also different from its conformation in the ACN free solution. Figure 3A shows the retention time and solution conformation energy of these peptides, and it was found that the retention order of these peptides in the C18 column was exactly the same as the order of their conformation energies. It can be determined that the conformation energy of DW11 was the highest in the 30% (v/v) ACN solution. The peptide ILK7F89 had the second highest conformation energy, followed by peptides DW4 and DK7. The peptide DL2 had the lowest conformation energy. The relationship between the retention time and conformation energy of peptides in pure water is shown in Figure 3B. The conformation energy of DW11 was still the highest. Interestingly, we found that the peptide DW4 had the second highest conformation energy, followed by the virgin IL-K7F89. Also, the conformation energy of peptide DL2 was higher than that of DK7 in the ACN free solution. It could be observed that the order of conformation energy altered as the solvent changed from the 30% ACN to ACN free solution. Furthermore, we have found that the order of conformation energy in water was again exactly the same as the retention order of peptides in the C18 column when the samples were prepared in 0% (v/v) ACN. From Tables 2 and 3, we found that the retention order of peptide diastereomers in RPC is well related to the peptide’s solution conformation energy; that is, the structural stability of peptide in solution could be an important indication on their retention in the RPC column, especially for the retention prediction of peptide diastereomers in RPC. We further estimated the intramolecular hydrophobic interaction by calculating the van der Waals energy among the hydrophobic residues in peptides, i.e., Ile1, Leu2, Trp4,

(2)

The nonbonded energy derives from intramolecular van der Waal’s attraction, electrostatic interaction, and hydrogen bonding. The bonded energy is obtained by summation of the bond, angle, and dihedral energies. The bonded and nonbonded energies of IL-K7F89 peptide diastereomers were estimated by all-atom molecular dynamics simulation. The AMBER force field was used to simulate peptide structure and estimate its conformation energy. In this study, each modeling system was performed for at least 10 ns, and all 13604

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other possible mechanism is the adsorption entropy effect. The increase in peptide−resin interaction does not necessarily reduce the adsorption enthalpy. As the hydrophobic contact increases, the interaction enthalpy is reduced, but the increased adsorption enthalpy is needed to repel the bound water. The reduction of adsorption free energy is not due to the reduction in adsorption enthalpy but results from the increase in desolvation entropy. The measurement of thermodynamic properties is necessary to investigate what causes the lower conformation energy diastereomer eluted earlier. Therefore, the adsorption enthalpy was obtained by measuring the retention factors at various temperatures. The relationship between the capacity factor and the equilibrium constant (Keq) are expressed by eq 3:

k′ = Keqϕ

(3)

where tR and t0 are the retention time of the peptide and nonretained molecule in the RPC column, respectively. The phase ratio, denoted by ϕ, is the ratio of stationary phase volume to mobile phase volume in the column. The standard adsorption free energy (ΔG°) of peptides transferred from the mobile phase to the stationary phase can be calculated by ΔG° = ΔH ° − T ΔS ° = −RT ln Keq = −RT ln k′ + RT ln ϕ

(4)

With the combination of eqs 3 and 4, the standard adsorption enthalpy (ΔH°) and entropy (ΔS°) can be calculated by eq 5 ln k′ =

Figure 3. Retention time and solution conformation energy of ILK7F89 peptide and its diastereomers by 30% ACN/H2O elution. The sample loading solvents are (A) 30% ACN/H2O and (B) 100% H2O, respectively.

−ΔH ° ΔS ° + + ln φ RT R

(5)

Isocratic elution of peptides in 30% (v/v) ACN at the temperature ranging from 20 to 30 °C was carried out to obtain the retention factors, k′. Figure 4 showed the linear van’t Hoff plots describing ln k′ as a function of 1/T. The adsorption enthalpies, ΔH° could therefore be calculated from the slopes according to eq 5. The Gibbs free energy of adsorption, ΔG°, was calculated directly from the retention factors, k′, according to eq 4 and the adsorption entropies could then be calculated.

Trp6, Phe8, Phe9, and Trp11. Tables 2 and 3 list the van der Waals energy among these residues calculated by the analysis of MD simulation. It was observed that the peptide exhibiting the lower conformation energy in sample loading solvent had a stronger van der Waals interaction among its hydrophobic residues. The above results support our previous hypothesis that the structure flexibility of peptides in solution indeed plays an important role in their retention behaviors in RP-HPLC.25 A structurally rigid peptide with the lower conformation energy is more stable in the elution solvent; thus, it is relatively unfavorable for the peptide to partition to the hydrophobic resin. Thermodynamics Analysis of Peptide Diastereomers and Hydrophobic Ligand Interaction. It is still unclear exactly how the conformation energy in solution affects peptide retention in a hydrophobic column. First, one possible mechanism is the adsorption enthalpy effect. The peptide with the higher conformation energy has a rather flexible structure in solution. Little deformation enthalpy is paid for increasing peptide−resin interaction. The lower overall adsorption enthalpy is obtained from either the lower deformation enthalpy or the lower interaction enthalpy. The peptide with higher conformation energy has a lower adsorption enthalpy and consequently a lower adsorption free energy. However, we have found in the previous study that the peptide isomer with the higher conformation energy in solution did not always have the lower adsorption enthalpy. Second, the

Figure 4. Linear van’t Hoff plots of IL-K7F89 peptide and its diastereomers. Both the sample loading and elution solvent are 30% ACN/H2O. 13605

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The obtained thermodynamics properties, ΔG°, ΔH°, and TΔS°, were plotted as shown in Figure 5. The results indicated

Figure 6. Relationship between the number of ordered water molecules near the IL-K7F89 peptide and its diastereomers and their solution conformation energy from the calculations of MD simulations of peptide diastereomers in 30% ACN/H2O.

Figure 5. Thermodynamic parameters of peptide and hydrophobic resin interaction. The sample loading and elution solvents are 30% ACN/H2O.

solution conformation energy was in accordance with the number of ordered waters surrounding the peptide. It meant that the peptide, which was more stable in aqueous phase, was surrounded by fewer order water molecules. This finding may explain why the retention order of these peptides in C18 column is related to their conformation energies in solution. The intramolecular hydrophobic interaction reduced the conformation energy as well as the exposed hydrophobic area. As the exposed hydrophobic area was reduced, the associated ordered water also decreased. Subsequently, the entropy gain from repelling ordered water was also reduced. As listed in Table 2, it was observed that the peptide exhibiting the lower conformation energy in the sample solvent had a stronger van der Waals interaction among its hydrophobic residues. The result was in accordance with the calculated number of ordered water molecules near peptides. The stronger van der Waals interaction among hydrophobic residues rendered a smaller exposed hydrophobic area of the peptide. Subsequently, the number of associated ordered water molecules decreased. These results reveal how the conformation energy in sample solvent affected the retention order of peptide diastereomers in a C18 column. The stronger van der Waals interaction among hydrophobic residues reduced the overall conformation energy. Simultaneously, the stronger interaction among hydrophobic residues reduced the number of associated ordered water molecules and resulted in lower desolvation entropy upon peptide adsorption. Subsequently the overall adsorption free energy increased and the column retention decreased. The adsorption entropy determined retention order observed from the thermodynamic measurements can therefore be explained.

that the adsorption enthalpies and entropies were all negative. This indicated that the adsorptions of all the peptides on C18 resins were driven by adsorption enthalpy. However, apparently the elution order was not determined by adsorption enthalpy. A lower adsorption enthalpy did not guarantee a longer retention. On the contrary, we found that the retention times correlated well with the adsorption entropies. The finding indicated that the retention order of IL-K7F89 diastereomers on C18 columns was determined by their adsorption entropies. The adsorption entropy was composed of peptide deformation entropy, desolvation entropy of associated solvents, and direct interaction entropy of the peptide to the solid phase.27 Among these three types of entropies, only the desolvation entropy was strongly positive. We considered that it was the adsorption entropy gains from repelling the ordered water molecules or associated ACN molecules near the hydrophobic residues of peptides that determined the retention order. Role of Associated Solvent on the Conformation Energy of Peptide Diastereomers. To understand the role of associated solvent on the retention of peptide diastereomers in the RPC column, we further evaluated the number of ordered water and associated ACN molecules around the peptide through the help of the all-atom MD simulation. In order to calculate the amount of ordered water molecules around a peptide, we evaluated the H-bond between water molecules near each peptide (the O atom of water within 8 Å from the peptide). The ordered water was defined by the number of H-bonds per water. The water molecule forming 3 H-bonds with neighboring waters was defined as an ordered water molecule. The region of 8 Å surrounding a peptide contained roughly two layers of water molecules around the hydrophobic residue, and the 3 H-bonds were defined by taking the value of the icelike water, which normally forms 3 to 4 Hbonds. From the last 2 ns of simulation results of peptide in 30% ACN/H2O, the average number of ordered water molecules was listed in Table 2. It was found that there were about 25−36 ordered water molecules around each peptide. We observed that the peptide with lower conformation energy was surrounded by less ordered water (Figure 6). The order of

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CONCLUSIONS We hypothesized in our previous study that the retention order of sequence shuffled peptide isomers could be predicted by their conformation energy in solution. In this study, we found that the retention order of peptide diastereomers could also be predicted through calculating their conformation energy in solution by all-atom MD simulation, and the composition of sample loading solvent would affect their conformations as well 13606

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as their retention order in a C18 column. Thermodynamic analysis revealed that the adsorption of these peptide diastereomers was driven by adsorption enthalpy but that the retention order was determined by the overall adsorption entropy. To understand the connections among retention time, conformation energy, and adsorption entropy, all-atom MD simulation was performed to calculate the intramolecular hydrophobic interaction of peptides and the number of ordered water molecules associated with them. It was found that the both intramolecular hydrophobic interaction and the number of associated ordered water molecules were well correlated with the adsorption entropy, the conformation energy, and the retention order. The relationship between conformation energy and retention order of these peptide diastereomers can be explained as follows: the intramolecular hydrophobic interaction reduces the conformation energy as well as the exposed hydrophobic area of peptides. As the exposed hydrophobic area is reduced, the associated ordered water molecules are also decreased. Subsequently, the adsorption entropy gain from repelling ordered water molecules is reduced. The reduction of entropy increases the overall free energy of adsorption and thereby shortens the retention time.

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ASSOCIATED CONTENT

S Supporting Information *

This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*R. Chyu Ruaan: E-mail: [email protected]; fax, +886-3280-4341; phone, +886-3-4227151 ext. 34232. W. Yih Chen: Email: [email protected]; fax, +886-3-422-5258; phone, +886-3-4227151 ext. 34222. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors would like to thank the National Science Council of Taiwan for the financial support of this project. They also thank the National Center for High-Performance Computing of Taiwan and the Vger computer cluster at the National Center University of Taiwan for providing computer time and facilities.

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REFERENCES

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