Reversed-phase liquid chromatography as a tool in the determination of the hydrophilicity/hydrophobicity of amino acid side-chains at a ligand-receptor interface in the presence of different aqueous environments. II. Effect of varying peptide ligand hydrophobicity

The present study represents a continuation of our development of a chromatographic model for studying the hydrophobic interactions which characterize the way a ligand binds to its receptor. We have designed 18-residue amphipathic alpha-helical peptides (representing the hydrophobic binding domain of a ligand), where the non-polar face interacts with the non-polar face of a reversed-phase stationary phase (representing a receptor protein with a hydrophobic binding pocket). Two series of amphipathic alpha-helical peptides were subjected to reversed-phase liquid chromatography at pH 2.0, where the "native" Ala-face peptide contains seven Ala residues in its non-polar face and the "native" Leu-face series contains seven Leu residues in its non-polar face. Mutants of the two series were then prepared by replacing one residue in the centre of the non-polar face in both series of peptides, resulting in amino acid side-chains being exposed to a moderately non-polar environment (Ala series) or a very hydrophobic environment (Leu series) surrounding the substitution site. With this model, we have demonstrated that an increase in non-polarity of the ligand enhances hydrophilicity (decreases hydrophobicity) of all amino acids at the ligand-receptor interface, this effect being dependent on the intrinsic hydrophilicity/hydrophobicity of the side-chain. The addition of salt to the aqueous environment surrounding the binding site of the ligand and receptor was also shown to affect the hydrophilicity/hydrophobicity of amino acids in the binding interface. For the Ala-face mutants, the majority of the non-polar side-chains and the three positively charged residues (Arg, His, Lys) showed significant enhancement of hydrophobicity in the presence of salt; in contrast, in the much more hydrophobic environment of the Leu-face mutants, there was a trend of lesser hydrophobicity enhancement and/or significantly more hydrophilicity enhancement in the presence of salt. Our results should have major implications for the understanding of the hydrophilicity/ hydrophobicity of side-chains in varying hydrophobic and aqueous environments.     

Reversed-phase liquid chromatography as a tool in the determination of the hydrophilicity/hydrophobicity of amino acid side-chains at a ligand-receptor interface in the presence of different aqueous environments. I. Effect of varying receptor hydrophobicity

We have developed further a chromatographic model for studying the hydrophobic interactions which characterize the way a ligand binds to its receptor. This model is based on observing the retention behaviour of de novo designed model 18-residue amphipathic alpha-helical peptides (representing the hydrophobic binding domain of a ligand) on reversed-phase packings by varying hydrophobicity (representing a receptor protein with a hydrophobic binding pocket). Mutants of the "native" peptide ligand (which contains seven Leu residues in its non-polar face) were designed by replacing one residue in the center of the extremely non-polar face of the amphipathic alpha-helix. Through reversed-phase liquid chromatography of these peptides at pH 2.0 on cyano and C18 columns, we have demonstrated how an increase in receptor hydrophobicity (represented by an increase in column stationary phase hydrophobicity; cyano --> C18) significantly enhances hydrophilicity of polar amino acid side-chains at the ligand-receptor interface while moderately enhancing the hydrophobicity of non-polar side-chains. The addition of salt (100 mM sodium perchlorate) to the aqueous environment surrounding the binding site of receptor and ligand was also shown to have a profound effect on side-chain hydrophilicity/hydrophobicity in the binding interface. This effect was particularly dramatic for the positively charged side-chains Arg, Lys and His, whose significant enhancement of hydrophobicity in the presence of the cyano column contrasted with their increase in hydrophilicity in the presence of the considerably more hydrophobic C18 stationary phase. Our results have major implications to understanding the influence of hydrophobic and aqueous environment on hydrophilicity/hydrophobicity of amino acid side-chains and the role side-chains play in the folding and stability of proteins.     

Effect of anionic ion-pairing reagent concentration (1-60 mM) on reversed-phase liquid chromatography elution behaviour of peptides

The homologous series of volatile perfluorinated acids-trifluoroacetic acid (TFA), pentafluoropropionic acid (PFPA) and heptafluorobutyric acid (HFBA)--continue to be excellent anionic ion-pairing reagents for reversed-phase high-performance liquid chromatography (RP-HPLC) after more than two decades since their introduction to this field. It was felt that a thorough, step-by-step re-examination of the effects of anionic ion-pairing reagents over a wide concentration range on RP-HPLC peptide elution behaviour is now due, particularly considering the continuing dominance of such reagents for peptide applications. Thus, RP-HPLC was applied over a range of 1-60 mM phosphoric acid, TFA, PFPA and HFBA to two mixtures of 18-residue synthetic peptides containing either the same net positive charge (+4) or varying positive charge (+1, +2, +3, +4). Peptides with the same charge are resolved very similarly independent of the ion-pairing reagent used, although the overall retention times of the peptides increase with increasing hydrophobicity of the anion: phosphate < TFA- < PFPA- < HFBA-. Peptides of differing charge move at differing rates relative to each other depending on concentration of ion-pairing reagents. All four ion-pairing reagents increased peptide retention time with increasing concentration, albeit to different extents, again based on hydrophobicity of the anion, i.e., the more hydrophobic the anion, the greater the increase in peptide retention time at the same reagent concentration. Interestingly, phosphoric acid produced the best separation of the four-peptide mixture (+1 to +4 net charge). In addition, concentrations above 10 mM HFBA produced a reversal of the elution order of the four peptides (+1 < + 2 < + 3 < + 4) compared to the elution order produced by the other three reagents over the entire concentration range (+4 < + 3 < + 2 < + 1).     

Effect of anionic ion-pairing reagent hydrophobicity on selectivity of peptide separations by reversed-phase liquid chromatography

Despite the continuing dominance of trifluoroacetic acid (TFA) as the anionic ion-pairing reagent of choice for peptide separations by reversed-phase high-performance liquid chromatography (RP-HPLC), we believe that a step-by-step approach to re-examining the relative efficacy of TFA compared to other ion-pairing reagents is worthwhile, particularly for the design of separation protocols for complex peptide mixtures, e.g., in proteomics applications. Thus, we applied RP-HPLC in the presence of different concentrations of anionic ion-pairing reagents - phosphoric acid, TFA, pentafluoropropionic acid (PFPA) and heptafluorobutyric acid (HFBA)--to a mixture of three groups of four 10-residue peptides, these groups containing peptides of +1, +3 or +5 net charge. Overall separation of the 12-peptide mixture improved with increasing reagent hydrophobicity (phosphate- < TFA- < PFPA- < HFBA-) and/or concentration of the anion, with reagent hydrophobicity having a considerably more pronounced effect than reagent concentration. HFBA, in particular, achieved an excellent separation at a concentration of just 10 mM, whereby the peptides were separated by charged groups (+1 < +3 < +5) and hydrophobicity within these groups. There was an essentially equal effect of reagent hydrophobicity and concentration on each positive charge of the peptides, a useful observation for prediction of the effect of varying counterion concentration hydrophobicity and/or concentration during optimization of peptide purification protocols. Peak widths were greater for the more highly charged peptides, although these could be decreased significantly by raising the acid concentration; concomitantly, peptide resolution increased with increasing concentration of ion-pairing reagent.     

Defining intrinsic hydrophobicity of amino acids' side chains in random coil conformation. Reversed-phase liquid chromatography of designed synthetic peptides vs. random peptide data sets

The two leading RP-HPLC approaches for deriving hydrophobicity values of amino acids utilize either sets of designed synthetic peptides or extended random datasets often extracted from proteomics experiments. We find that the best examples of these two methods provide virtually identical results--with exception of Lys, Arg, and His. The intrinsic hydrophobicity values of the remaining residues as determined by Kovacs et al. (Biopolymers 84 (2006) 283) correlates with an R(2)-value of 0.995+ against amino acid retention coefficients from our Sequence Specific Retention Calculator model (Anal. Chem. 78 (2006) 7785). This novel finding lays the foundation for establishing consensus amino acids hydrophobicity scales as determined by RP-HPLC. Simultaneously, we find the assignment of hydrophobicity values for charged residues (Lys, Arg and His at pH 2) is ambiguous; their retention contribution is strongly affected by the overall peptide hydrophobicity. The unique behavior of the basic residues is related to the dualistic character of the RP peptide retention mechanism, where both hydrophobic and ion-pairing interactions are involved. We envision the introduction of "sliding" hydrophobicity scales for charged residues as a new element in peptide retention prediction models. We also show that when using a simple additive retention prediction model, the "correct" coefficient value optimization (0.98+ correlation against values determined by synthetic peptide approach) requires a training set of at least 100 randomly selected peptides.     

Capillary electrophoresis of cationic random coil peptide standards: effect of anionic ion-pairing reagents and comparison with reversed-phase chromatography

The present study compares a charge/hydrophobicity capillary electrophoresis (CE) approach to reversed-phase high-performance liquid chromatography (RP-HPLC) for the separation of three series of four synthetic, random coil peptide standards. Each series has peptides of the same positive charge (+1, +2 and +3 series) and length but differing in hydrophobicity. Complete resolution of the 12 peptides was achieved via a novel CE approach: a capillary zone electrophoresis (CZE) mode effected a separation of identically charged peptides; within each charged group of peptides, the addition of perfluorinated acid anionic ion-pairing reagents allowed resolution of the peptides through a mechanism based on peptide hydrophobicity which we have termed ioninteraction (II)-CZE. The peak capacity and peptide resolution of this CE approach was superior to that of RP-HPLC and stresses an important role for CE for peptide/proteomic applications.     

Requirements for prediction of peptide retention time in reversed-phase high-performance liquid chromatography: hydrophilicity/hydrophobicity of side-chains at the N- and C-termini of peptides are dramatically affected by the end-groups and location

The value of reversed-phase high-performance liquid chromatography (RP-HPLC) and the field of proteomics would be greatly enhanced by accurate prediction of retention times of peptides of known composition. The present study investigates the hydrophilicity/hydrophobicity of amino acid side-chains at the N- and C-termini of peptides while varying the functional end-groups at the termini. We substituted all 20 naturally occurring amino acids at the N- and C-termini of a model peptide sequence, where the functional end-groups were N(alpha)-acetyl-X- and N(alpha)-amino-X- at the N-terminus and -X-C(alpha)-carboxyl and -X-C(alpha)-amide at the C-terminus. Amino acid coefficients were subsequently derived from the RP-HPLC retention behaviour of these peptides and compared to each other as well as to coefficients determined in the centre of the peptide chain (internal coefficients). Coefficients generated from residues substituted at the C-terminus differed most (between the -X-C(alpha)-carboxyl and -X-C(alpha)-amide peptide series) for hydrophobic side-chains. A similar result was seen for the N(alpha)-acetyl-X- and N(alpha)-amino-X- peptide series, where the largest differences in coefficient values were observed for hydrophobic side-chains. Coefficients derived from substitutions at the C-terminus for hydrophobic amino acids were dramatically different compared to internal coefficients for hydrophobic side-chains, ranging from 17.1 min for Trp to 4.8 min for Cys. In contrast, coefficients derived from substitutions at the N-terminus showed relatively small differences from the internal coefficients. Subsequent prediction of peptide retention time, within an error of just 0.4 min, was achieved by a predictive algorithm using a combination of internal coefficients and coefficients for the C-terminal residues. For prediction of peptide retention time, the sum of the coefficients must include internal and terminal coefficients.     

Ion-interaction CZE: the presence of high concentrations of ion-pairing reagents demonstrates the complex mechanisms involved in peptide separations

We have furthered our understanding of the separative mechanism of a novel CE approach, termed ion-interaction CZE (II-CZE), developed in our laboratory for the resolution of mixtures of cationic peptides. Thus, II-CZE and RP-HPLC were applied to the separation of peptides differing by a single amino acid substitution in 10- and 12-residue synthetic model peptide sequences. Substitutions differed by a wide range of properties or side-chain type (e.g., alkyl side-chains, polar side-chains, etc.) at the substitution site. When carried out in high concentrations (400 mM) of pentafluoropropionic acid (PFPA), II-CZE separated peptides in order of increasing hydrophobicity when the substituted side-chains were of a similar type; when II-CZE was applied to the mixtures of peptides with substitutions of side-chains that differed in the type of functional group, there was no longer a correlation of electrophoretic mobility in II-CZE with relative peptide hydrophobicity, suggesting that a third factor is involved in the separative mechanism beyond charge and hydrophobicity. Interestingly, the hydrophobic PFPA- anion is best for separating peptides that differ in hydrophobicity with hydrophobic side-chains but high concentrations of the hydrophilic H2PO4- anion are best when separating peptides that differ in polar side-chains relative to hydrophobic side-chains. We speculate that differential hydration/dehydration properties of various side-chains in the peptide and the hydration/dehydration properties of the hydrophilic/hydrophobic anions as well as the electrostatic attractions between the peptide and the anions in solution all play a critical role in these solution-based effects.     

Quantitation of the nearest-neighbour effects of amino acid side-chains that restrict conformational freedom of the polypeptide chain using reversed-phase liquid chromatography of synthetic model peptides with L- and D-amino acid substitutions

Side-chain backbone interactions (or "effects") between nearest neighbours may severely restrict the conformations accessible to a polypeptide chain and thus represent the first step in protein folding. We have quantified nearest-neighbour effects (i to i+1) in peptides through reversed-phase liquid chromatography (RP-HPLC) of model synthetic peptides, where L- and D-amino acids were substituted at the N-terminal end of the peptide sequence, adjacent to a L-Leu residue. These nearest-neighbour effects (expressed as the difference in retention times of L- and D-peptide diastereomers at pHs 2 and 7) were frequently dramatic, depending on the type of side-chain adjacent to the L-Leu residue, albeit such effects were independent of mobile phase conditions. No nearest-neighbour effects were observed when residue i is adjacent to a Gly residue. Calculation of minimum energy conformations of selected peptides supported the view that, whether a L- or D-amino acid is substituted adjacent to L-Leu, its orientation relative to this bulky Leu side-chain represents the most energetically favourable configuration. We believe that such energetically favourable, and different, configurations of L- and D-peptide diastereomers affect their respective interactions with a hydrophobic stationary phase, which are thus quantified by different RP-HPLC retention times. Side-chain hydrophilicity/hydrophobicity coefficients were generated in the presence of these nearest-neighbour effects and, despite the relative difference in such coefficients generated from peptides substituted with L- or D-amino acids, the relative difference in hydrophilicity/hydrophobicity between different amino acids in the L- or D-series is maintained. Overall, our results demonstrate that such nearest-neighbour effects can clearly restrict conformational space of an amino acid side-chain in a polypeptide chain.     

Ion-interaction-capillary zone electrophoresis of cationic proteomic peptide standards

We have employed a novel capillary electrophoresis (CE) approach recently developed in our laboratory, termed ion-interaction-capillary zone electrophoresis (II-CZE), to the resolution of a mixture of 27 synthetic cationic proteomic peptide standards. These peptides were comprised of three groups of nine peptides (with net charges of +1, +2 and +3 for all nine peptides within a group), the hydrophobicity of the nine peptides within a group varying only subtly between adjacent peptides. This bidimensional CE approach achieved excellent resolution of the peptides with high peak capacity by combining the powerful CZE mechanism located in the background electrolyte (BGE) with an hydrophobicity-based mechanism also located in the BGE, the latter consisting of high concentrations (up to 0.4M) of aqueous perfluorinated acids (trifluoroacetic acid, pentafluoropropionic acid and heptafluorobutyric acid). Thus, concomitant with a CZE separation of the three differently charged groups of peptides, there is an hydrophobically-mediated separation of the peptides within these groups effected through interaction of the hydrophobic anions of the perfluorinated acids with hydrophobic amino acid side-chains in the peptides. This methodology is dramatically different from other CE methods that have used complexing agents such as micelles or cyclodextrins in MEKC. Overall, the results presented here demonstrate the value of CE as a peptide separative tool in its own right, including its use for proteomic applications, and not merely as a complementary technique to reversed-phase high-performance liquid chromatography (RP-HPLC).     

The perchlorate anion is more effective than the trifluoroacetate anion as an ion-pairing reagent for reversed-phase chromatography of peptides

The addition of salts, specifically sodium perchlorate (NaClO4), to mobile phases at acidic pH as ion-pairing reagents for reversed-phase high-performance liquid chromatography (RP-HPLC) has been generally overlooked. To demonstrate the potential of NaClO4 as an effective anionic ion-pairing reagent, we applied RP-HPLC in the presence of 0-100 mM sodium chloride (NaCl), sodium trifluoroacetate (NaTFA) or NaClO4 to two mixtures of synthetic 18-residue peptides: a mixture of peptides with the same net positive charge (+4) and a mixture of four peptides of +1, +2, +3 and +4 net charge. Interestingly, the effect of increasing NaClO4 concentration on increasing peptide retention times and selectivity changes was more dramatic than that of either NaCl or NaTFA, with the order of increasing anion effectiveness being Cl- < TFA- < C104-. Such effects were more marked when salt addition was applied to eluents containing 10 mM phosphoric acid (H3PO4) compared to 10 mM trifluoroacetic acid (TFA) due to the lesser starting anion hydrophobicity of the former mobile phase (containing the phosphate ion) compared to the latter (containing the TFA- ion).     

Selectivity differences in the separation of amphipathic alpha-helical peptides during reversed-phase liquid chromatography at pHs 2.0 and 7.0: effects of different packings, mobile phase conditions and temperature

In an ongoing effort to understand the effect of varying reversed-phase high-performance liquid chromatography (RP-HPLC) parameters on the retention behaviour of peptides, necessary for the rational development of separation/optimization protocols, we believe it is important to delineate the contribution of alpha-helical structure to the selectivity of peptide separations. The present study reports the effects of varying column packing, mobile phase conditions and temperature on RP-HPLC retention behaviour at pHs 2.0 and 7.0 of peptides based on the amphipathic peptide sequence Ac-EAEKAAKEXEKAAKEAEK-amide (with position X in the centre of the hydrophobic face of the alpha-helix), where position X is substituted by L- or D-amino acids. At pH 2.0, an increase in trifluoroacetic acid concentration or the addition of sodium perchlorate to a phosphoric acid-based mobile phase had the similar effect of improving peak shape as well as increasing peptide retention time due to ion-pairing effects with the positively-charged peptides; in contrast, at pH 7.0, the addition of salt had little effect save an improvement in peak shape. Temperature was shown to have a complex influence on peptide selectivity due to varying effects on peptide conformation. In addition, subtle effects on peptide selectivity were also noted based on the column packings employed at pHs 2.0 and 7.0.     

Mixed-mode hydrophilic interaction/cation-exchange chromatography: separation of complex mixtures of peptides of varying charge and hydrophobicity

Mixed-mode hydrophilic interaction/cation-exchange chromatography (HILIC/CEX) was applied to the separation of two mixtures of synthetic peptide standards: (i) a 27-peptide mixture containing three groups of peptides (each group containing nine peptides of the same net charge of +1, +2 or +3), where the hydrophilicity/hydrophobicity of adjacent peptides within the groups varied only subtly (generally by only a single carbon atom); and (ii) peptide pairs with the same composition but different sequences, where the sole difference between the peptides was the position of a single amino acid substitution. HILIC/CEX is essentially CEX chromatography in the presence of high levels of organic modifier (generally ACN). The present study demonstrated the dramatic effect of increasing ACN concentration (optimum levels of 60-80%, depending on the application) on the separation of both mixtures of peptides. The greater the charge on the peptides, the better the separation achievable by HILIC/CEX. In addition, HILIC/CEX separation of both the peptide mixtures used in the present study was shown to be superior to that of the more commonly applied RP-HPLC mode. Our results highlight again the efficacy of HILIC/CEX as a peptide separation mode in its own right as well as an excellent complement to RP-HPLC.     

Quantitative electrospray ionization mass spectrometric studies of ternary complexes of amino acids with Cu(2+) and phenanthroline

Electrospray ionization (ESI) mass spectra of ternary complexes of Cu(2+) and 1,10-phenanthroline with the 20 essential amino acids (AA) were investigated quantitatively. Non-basic amino acids formed singly charged complexes of the [Cu(AA - H)phen](+) type. Lysine (Lys) and arginine (Arg) formed doubly charged complexes of the [Cu(HAA - H)phen](2+) type. Detection limits were determined for the complexes of phenylalanine (Phe), glutamic acid (Glu) and Arg, which were at low micromolar or submicromolar concentrations under routine conditions. Detection limits of low nanomolar concentrations are possible for amino acids with hydrophobic side-chains (Phe, Tyr, Trp, Leu, Ile) as determined for Phe. The efficiencies for the formation by ESI of gaseous [Cu(AA - H)phen](+) ions were determined and correlated with the acid-base properties of the amino acids, ternary complex stability constants and amino acid hydrophobicities expressed as the Bull-Breese indices (DeltaF). A weak correlation was found between DeltaF and the ESI efficiencies for the formation of gaseous [Cu(AA - H)phen](+) [Cu(HAA - H]phen](2+) and [AA + H](+) ions that showed that amino acids with hydrophobic side-chains were ionized more efficiently. In the ESI of binary and ternary amino acid mixtures, the formation of gas-phase Cu-phen complexes of amino acids with hydrophobic side-chains was enhanced in the presence of complexes of amino acids with polar or basic side-chains. An interesting enhancement of the ESI formation of [Cu(Glu - H)phen](+) was observed in mixtures. The effect is explained by ion-cluster formation at the droplet interface that results in enhanced desorption of the glutamic acid complex.     

Essential of Proline and Valine Residues in the Peptide Derived from Lactoferrin for Angiotensin Converting Enzyme Inhibition

We synthesized Leu‐Arg‐Pro‐Val‐Ala‐Ala‐Glu, the peptide contained in lactoferrin (Lf), to identify the angiotensin converting enzyme (ACE) inhibition. In an attempt to know the structure‐activity relationship of this peptide, we replaced Pro (the third amino acid residues from N‐terminal) or Val (the fourth amino acid residues from N‐terminal) with Ala (neutral amino acid), Glu (acidic amino acid) or Lys (basic amino acid) to produce six peptides. From the in vitro ACE inhibition (IC₅₀) of these synthesized peptides, the original peptide (Leu‐Arg‐Pro‐Val‐Ala‐Ala‐Glu) showed higher ACE inhibition than the replaced six peptides. Thus, replacement of Pro at the third amino acid residues or Val at the fourth position with Ala, Glu or Lys revealed the ACE inhibition to be lower than the original form of Leu‐Arg‐Pro‐Val‐Ala‐Ala‐Glu. Otherwise, we added one peptide at the C‐terminal of Leu‐Arg‐Pro‐Val‐Ala‐Ala‐Glu and found both products with an addition of Val (Leu‐Arg‐Pro‐Val‐Ala‐Ala‐Glu‐Val) or Ile (Leu‐Arg‐Pro‐Val‐Ala‐Ala‐Glu‐Ile) showing a lower ACE inhibition than the original one. The ACE inhibitions produced by both replaced peptides were without significance. Also, deletion of the last peptide at the C‐terminal (Leu‐Arg‐Pro‐Val‐Ala‐Ala) failed to produce a marked change of ACE inhibition as compared to the original one. These results suggest that Pro and Val are essential in the peptide for inhibition of ACE activity.     

Retention behavior of peptides in hydrophilic-interaction chromatography

Selected hydrophilic interaction chromatography (HILIC) columns packed with bare silica, bridge-ethyl hybrid silica, or an amide sorbent chemistry were utilized for an investigation of chromatographic behavior and separation selectivity of tryptic peptides. Retention model was proposed allowing for retention prediction of peptides with correlation coefficient R(2)~0.92-0.97 for various columns. The values of optimized amino acid retention coefficients were compared to those obtained for reversed-phase liquid chromatography (Gilar et al., Anal. Chem. 2010, 82, 265-275) and used to elucidate the impact of different amino acid on peptide HILIC retention. In contrast to reversed-phase chromatography, where presence of Phe, Trp, Ile, and Leu amino acid residues in sequence strongly promoted, and presence of hydrophilic His, Lys and Arg residues strongly reduced peptide retention, the effects of these amino acid residues in HILIC were opposite (His, Lys and Arg promote, Phe, Trp, Ile and Leu demote peptide retention in HILIC). Retention coefficient optimized for pH experiments illustrated the impact of silanols on HILIC retention.     

Reversed-phase HPLC of peptides: Assessing column and solvent selectivity on standard, polar-embedded and polar endcapped columns

We desired to evaluate the chromatographic selectivity for peptides of silica-based RP high-performance liquid chromatography stationary phases with various modifications (polar embedding and polar endcapping on C(18) columns; ether-linked phenyl column with polar endcapping) compared with n-alkyl (C(18), C(8)) and aromatic phenylhexyl columns. Thus, we have designed and synthesized two series of synthetic peptide standards with the sequence Gly-Gly-Leu-Gly-Gly-Ala-Leu-Gly-X-Leu-Lys-Lys-amide, where the N-terminal either contains a free α-amino group (AmC series) or is N(α)-acetylated (AcC series) and where position X is substituted by Gly, Ala, Val, Ile, Phe or Tyr. These represent series of peptides with single substitutions of n-alkyl (Gly相似文献   

Capillary electrophoresis of synthetic peptide standards varying in charge and hydrophobicity

A mixture of eight structurally closely related synthetic peptides as capillary electrophoretic (CE) standards is introduced. The almost identical mass-to-charge ratio of the standards, coupled with their random-coil (i.e., no secondary structure) nature, offer a potent analytical test for CE to separate peptides varying only subtly in hydrophobicity. Parameters varied to effect a separation included background electrolyte concentration, temperature, applied voltage in capillary zone electrophoresis (CZE in uncoated capillaries), as well as the introduction of hydrophobic mechanisms to the separation either through the use of micelles or C8-coated capillaries. Our step-by-step approach culminated in an optimized combination of a CZE mechanism for separation of differently charged peptide groups (based on common mass-to-charge ratio) and an ion-pairing mechanism (effecting a separation within each group of identically charged peptides), which we have termed ion-interaction CZE or II-CZE. The study clearly shows how the peptide standards allow an excellent assessment of the resolving power of CE.     

Host-guest chemistry in the gas phase: complex formation with 18-crown-6 enhances helicity of alanine-based peptides

The gas-phase helix propensities of alanine-based polypeptides are studied with different locations of a Lys residue and host-guest interactions with 18-Crown-6 (18C6). A series of model peptides Ac-Ala(9-n)-LysH(+)-Ala(n) (n = 0, 1, 3, 5, 7, and 9) is examined alone and with 18C6 using traveling wave ion mobility mass spectrometry combined with molecular dynamics (MD) simulations. The gas-phase helices are observed from the peptides whose Lys residue is located close to the C-terminus so that the Lys exerts its capping effect on the C-terminal carbonyl groups. The peptides, which interact with 18C6 in the gas phase, show enhanced helical propensities. The enhanced helicity of the peptide in the complex is attributed by isolation of the Lys butylammonium group from the helix backbone and the interaction of methylene groups of 18C6, which possess localized positive partial charges, with C-terminal carbonyl groups serving as a cap to stabilize the helix.     

Cytotoxic potency of small macrocyclic knot proteins: structure-activity and mechanistic studies of native and chemically modified cyclotides

The cyclotides are a family of circular and knotted proteins of natural origin with extreme enzymatic and thermal stability. They have a wide range of biological activities that make them promising tools for pharmaceutical and crop-protection applications. The cyclotides are divided into two subfamilies depending on the presence (M?bius) or absence (bracelet) of a cis-Pro peptide bond. In the current work we report a series of experiments to give further insight into the structure-activity relationship of cyclotides in general, and the differences between subfamilies and the role of their hydrophobic surface in particular. Selective chemical modifications of Glu, Arg, Lys and Trp residues was tested for cytotoxic activity: derivatives in which the Trp residue was modified showed low effect, demonstrating the existence of a connection between hydrophobicity and activity. However, over the full set of cyclotides examined, there was no strong correlation between the cytotoxic activity and their hydrophobicity. Instead, it seems more like that the distribution of charged and hydrophobic residues determines the ultimate degree of potency. Furthermore, we found that while the Glu residue is very important in maintaining the activity of the bracelet cyclotide cycloviolacin O2, it is much less important in the M?bius cyclotides. Despite these differences between cyclotide subfamilies, a systematic test of mixtures of cyclotides revealed that they act in an additive way.