Effect of cyclization of N-terminal glutamine and carbamidomethyl-cysteine (residues) on the chromatographic behavior of peptides in reversed-phase chromatography

N-terminal loss of ammonia is a typical peptide modification chemical artifact observed in bottom-up proteomics experiments. It occurs both in vivo for N-terminal glutamine and in vitro following enzymatic cleavage for both N-terminal glutamine and cysteine alkylated with iodoacetamide. In addition to a mass change of −17.03 Da, modified peptides exhibit increased chromatographic retention in reversed-phase (RP) HPLC systems. The magnitude of this increase varies significantly depending on the peptide sequence and the chromatographic condition used. We have monitored these changes for extensive sets (more than 200 each) of tryptic Gln and Cys N-terminated species. Peptides were separated on 100 Å pore size C18 phases using identical acetonitrile gradient slopes with 3 different eluent compositions: 0.1% trifluoroacetic acid; 0.1% formic acid and 20 mM ammonium formate at pH 10 as ion-pairing modifiers. The observed effect of this modification on RP retention is the product of increased intrinsic hydrophobicity of the modified N-terminal residue, lowering or removing the effect of ion-pairing formation on the hydrophobicity of adjacent residues at acidic pHs; and possibly the increased formation of amphipathic helical structures when the positive charge is removed. Larger retention shifts were observed for Cys terminated peptides compared to Gln, and for smaller peptides. Also the size of the retention increase depends on the eluent conditions: pH 10 ? trifluoroacetic acid < formic acid. Different approaches for incorporation these findings in the peptide retention prediction models are discussed.     

Peptide mapping with mobile phases of intermediate pH value using capillary reversed-phase high-performance liquid chromatography/electrospray ionisation tandem mass spectrometry

This investigation describes the separation of tryptic peptides by capillary reversed-phase high-performance liquid chromatography (RP-HPLC) with eluents in the intermediate pH range, followed by in-line electrospray ionisation tandem mass spectrometry (ESI-MS/MS) analysis. For these purposes, gradient elution procedures with an aqueous eluent containing 20 mM ammonium formate, and an increasing content of acetonitrile or methanol, were employed. Compared to the analysis of the same tryptic peptides under low-pH conditions with an ion-pairing reagent, the increase in the pH with the 20 mM ammonium formate mobile phase led to significant changes in both peptide retention to the reversed-phase column and the collision-induced dissociation at the MS/MS stage as a consequence of the changes in the physico-chemical properties of these peptides, such as their overall charge, polarity and relative hydrophobicity. Thus, improved selectivity for the peptide separation and favourable tandem mass spectrometry analysis could be obtained with eluents in this intermediate pH range. The number of tryptic peptides identified by the new approach for the proteins investigated were significantly higher than that obtained by the conventional low-pH methods. Moreover, analysis of protein digests at very low concentrations was also performed under both acidic and intermediate pH conditions and similar improvements in selectivity and MS/MS detection limits were observed, i.e. identification of more distinct peptides and higher sequence coverage of the protein was obtained when eluents of intermediate pH were employed. This study therefore highlights the potential of conducting peptide mapping in the intermediate pH range to achieve more reliable and sensitive protein identifications with capillary RP-HPLC–ESI-MS/MS.     

Ion-exchange properties of silica gel with covalently bonded histidine

The ion-exchange and acid-base properties of Silasorb 600 (10 μm) with immobilized histidine (His-SiO₂) were investigated. The acid-base properties of the sorbent were estimated from the dependence sof the retention of organic acids and bases on the eluent pH. The effect of various organic modifiers (benzoic, citric, oxalic, and sulfuric acids) and the eluent pH on the retention of inorganic ions was studied. It was shown that His-SiO₂ exhibits anion-exchange properties in acidic and weakly acidic media at pH < 5.7. Eight anions were separated under optimal conditions on a 150 × 4.6-mm column in 21 min using a 5 mM oxialic acid solution as an eluent.     

Effect of Substituents on the Retention in HPLC Chromatography of Strychnine Derivatives

Abstract

The capacity factork, relative retentions α_SP and log α_SP values measured on μPorasil columns for 33 strychnine derivatives using CHCl₃:MeOH (containing ca 2% NH₄OH) (93:7) as eluent in normal-phase chromatography. The results allow for the estimation of the effect of various substituents on the retention of these alkaloids.     

Reversed Phase High Performance Liquid Chromatography Using Carboxylic Acids in the Mobile Phase

Abstract

This report describes the use of different carboxylic acids as mobile phase modifiers. The effect on retention of acid chain length, pH, and eluent composition for a series of phenylalkanols, phenol, and the amines aniline, N-methylaniline, and benzylamine is discussed. The retention of both neutral and positively charged compounds is influenced by the dissociation equilibrium of the carboxylic acid in the mobile phase. By using l-pentanol to coat excess exposed silanol groups on the reversed phase column used, the inflection in the retention of both neutral and charged solutes as pH is changed occurs at the pK_a of the acid in the mobile phase. In addition, by using an acid and amine with the same or similar pK_a values, selective ion-pairing of this pair over others with dissimilar pK_a values can be promoted. Application of this technique to the selective retention of amino acids and peptides was unsuccessful.     

The pH Influence on the HPLC-Retention of Chemotherapeutic Fluoroquinolone Derivatives

Abstract

The gyrase inhibitor fluoroquinolone derivatives were investigated by RP-HPLC methods in C₁₈/methanol-phosphate buffer system. Considering the amphoteric feature of the substances it was reasonable to study the relationship between their chromatographic behaviour vs. pH of the mobil phase. The formation of k¹ values was characterized by a maximum curve, and the retention maxima could be found close to or at the pH values of isoelectric point of the compounds. This may be explained by the presence of the zwitter ionic or the unionized form of the derivatives at this pH value.

By addition of ion-pairing (IP) agent (cetrimide or hexanesulfonic acid) in different concentration to the eluent a definite k' value increase (formation of a characteristic saturation type curve) was observed indicating an ion-pairing mechanism.

In case of IPHPLC significant correlation was found between the octanol-water partition coefficients measured at the isoelectric point or at pH 7. 4, and the retention values of the compounds.     

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.     

Reductive Isopropylation of Amino Groups in Lysine Containing Peptides

Abstract

Model peptides, Gly-Gly-Lys-Arg, Arg-Lys-Asp-Val-Tyr, and Pro-Gly-Lys-Ala-Arg were reductively alkylated with [²H₆]acetone and sodium borohydride to assess the effects on peptide behavior. Lysine residues were converted to ?-N-isopropyllysine which eluted between phenylalanine and histidine on amino acid analysis. Amino terminal groups were also modified to an extent which depended on the particular peptide (glycine 100%, arginine 30%, and proline 10%-20%). High voltage paper electrophoresis of native and isopropylated peptides showed similar properties except for minor decreases in the mobility of the modified peptides due mainly to increased molecular weight. Isopropyllysine was not an effective substrate for trypsin, and α-N-isopropyl-amino acids did not form dansyl chloride derivatives. These findings should aid in the location, by peptide mapping techniques, of specific modified residues in reductively isopropylated proteins.     

Lipophilicity determination of some fully protected peptides and amino acids

Summary The lipophilicity of 21 fully protected peptides and amino acids was determined by reversed-phase thin-layer chromatography. The R_M values decreased linearly with growing methanol concentration of the eluent. The sequence and conformation of molecular substructures did not significantly influence the lipophilicity. The presence of salt and ammonia in the eluent had a negligible impact on retention; the effect of the pH value was also low. In the presence of 1M and 2M acetic acid the retention of each compound considerably decreased. Acetic acid also changed markedly the selectivity. Our data suggest that the acetic acid has a preponderant role in changing the retention and selectivity of fully protected peptides and amino acids in reversedphase thin-layer chromatography.     

Histidine as a dipolar eluent in ion chromatography of aliphatic amines

Summary Acidified L-histidine was found to be a suitable eluent in membrane-suppressed cation chromatography for the separation of aliphatic mono-amines (methyl, ethyl, trimethyl, triethyl, tripropylamines). Based on the protonation equilibrium of amines with the histidine eluent, a simplified separation method was developed, in which the eluent can act as a source of diprotic cations and analytes as monoprotic amines. On detection, the suppressor converts the eluent by deprotonation to its dipolar form with minimum conductivity at the pH of the isoelectric point of histidine, (pl 7.56). Efficient separations were observed under isocratic development with an eluent concentration of C_HIS≤2 mM and a pH below 2.0. The effect of eluent concentration and number of carbon atoms in the aliphatic chain on the retention and the resolution was determined. Selectivity data of the separation system are also presented. Presented at Balaton Symposium '01 on High-Performance Separation Methods, Siófok, Hungary, September 2–4, 2001     

Structure‐guided RP‐HPLC chromatography of diastereomeric α‐helical peptide analogs substituted with single amino acid stereoisomers

An α‐helical model peptide (Ac‐EAEKAAKE‐X‐EKAAKEAEK‐amide) was used as a template to examine the efficacy of conventional reversed‐phase high‐performance liquid chromatography (RP‐HPLC) in separating peptide analogs with single substitutions (at position X) of diasteromeric amino acids Ile, allo‐Ile, d ‐Ile and d ‐allo‐Ile. We compared differences in peptide retention behavior on a C₈ column and a C₁₈ column at different temperatures. We demonstrated how subtle differences in peptide secondary structure affected by the different substitutions of amino acids with identical overall hydrophobicity enabled effective resolution of these peptide analogs. We also demonstrated the ability of RP‐HPLC to separate Ile‐ and allo‐Ile‐substituted analogs of a 26‐residue α‐helical antimicrobial peptide (AMP), with the substitution site towards the C‐terminus of the α‐helix. These peptides show different values of antibacterial activity and hemolytic activity, and different selectivity against bacteria and human cells. Our results underline the ability of RP‐HPLC to resolve even difficult diasteromeric peptide mixtures as well as its value in monitoring very subtle hydrophobicity changes in de novo‐designed AMP. Copyright © 2013 John Wiley & Sons, Ltd.     

pH gradient reversed-phase liquid chromatography as a fractionation tool for the separation of peptides

Recent reports from our laboratory presented a comprehensive theory and demonstrated feasibility of reversed-phase liquid chromatography (RP-LC) employing the programmed gradient of pH of the mobile phase. The aim of that work was to explore the usefulness of the pH gradient-based approach in fractionation of peptides. The experiments were performed on a series of peptides separated at various LC conditions. Retention parameters of peptides in the pH gradient and in the simultaneous pH/organic modifier gradient RP-LC were compared. The best results were obtained with eluents comprising low but constant concentrations of organic modifier while gradient of pH in the mobile phase was developed several times during each chromatographic run. The elaborated LC conditions allowed controlling the elution of peptides not only according to their hydrophobic properties, but also taking into account their electronic properties, represented by isoelectric point (pI) values. The combination of isocratic (regarding organic modifier) LC mode with recurring eluent pH gradient is proposed as an effective fractionation method of peptide mixtures. Moreover, information on hydrophobicity and pI of the peptides, obtained by that approach, might be an additional peptides database matching constraint. Hence, a new tool for analytical and bioinformatics studies of peptides fractionation is proposed.     

Analysis of Group Retention Contributions for Peptides Separated by Reversed Phase High Performance Liquid Chromatography

Abstract

Within the framework provided by solvophobic theory, selectivities for unprotected peptides separated on fully porous, microparticulate, chemically bonded alkylsilicas can be ascribed to differences between the effective hydrophobic contact areas of the solutes. Furthermore, this theoretical treatment predicts that retention behaviour differences can be evaluated from topological parameters which accomodate the influence of amino acid side chain and end group contributions in the retention process. With data obtained for 57 peptides, including a variety of peptide hormones, eluted under the same conditions from a μBondapak C₁₈ column, these predictions have been rigorously tested using two methods of numerical analysis. The results provide further evidence that the hydrophobic group retention contributions of the amino acid residues in small peptides have an essentially additive effect on peptide retention with alkylsilicas. Divergences in retention behaviour are interpreted in terms of specific silanophilic and solvation interactions.     

Study of pH dependence using molecular modeling in reversed-phase liquid chromatography of 1-phenyl-3-methyl-5-pyrazolone derivatives of aldose

A series of 1-phenyl-3-methyl-5-pyrazolone (PMP) aldose was eluted on a C₁₈ column with an acetonitrile-phosphate buffer over the range of pH 2-10 in reversed phase liquid chromatography. The relationship between the retention factor, k, and the eluent pH was expressed by an equilibrium scheme representing the probable retention process. The experimental data were fitted to the correlation curve drawn according to the equation containing the formation of the hydrogen bond with a hydroxonium ion in the eluent and the ionization of the oxygen atoms in the pyrazolone rings of the solute.The conformational changes of PMP-aldose according with the eluent pH were corroborated by computer-aided molecular modeling using quantum chemical calculations. The retention behavior was elucidated as a function of the geometrical feature of the PMP-aldose.     

Retention,enantioselectivity and enantiomeric elution order of propranolol and its ester derivatives on an alpha1-acid glycoprotein-bonded column

Summary The retention, enantionselectivity and enantiomeric elution order of racemic propranolol (PP) and its ester derivatives (O-acetyl,-propionyl,-butyrul and-valeryl PP) on an ₁-acid glycoprotein (AGP)-bonded column have been investigated by changing eluent composition (eluent pH, buffer concentration, type and content of organic modifier). The retention of these cationic solutes, PP and its ester derivatives, was influenced by eluent pH, ionic strength and organic modifier content. The enantioselectivity was dependent on eluent pH and type of organic modifier. Reversal of the enantiomeric elution order of ester derivatives of PP (O-propionyl-butyryl) and-valeryl PP) occurred around eluent pH 6–7. These results suggst that chiral recognition or binding properties may be altered by the change in eluent composition, espeically eluent pH and type of organic modifier.     

Determination of pH value of isoelectric solution and identification of passive iron surface phases using immersion method

Using the method of phase modeling, the pH values of solutions corresponding to the uncharged surface of passive iron and ferric oxide γ-Fe₂O₃ (pH₀) are compared. According to the theory of connected places, the charge of metal oxide surface is determined by the adsorption or desorption of hydrogen ions leading to a change in the potential drop at the oxide/solution interface. Preliminarily passivated iron electrode was washed with twice-distilled water and placed into 0.5 M NaNO₃ solution with various pH values; the variation in the potential (ΔE) with time was studied. The pH₀ value for passive electrode under the open-circuit conditions was determined by the dependence of ΔE on the pH value (pH₀ 6.2 ± 0.1). The pH₀ value was close to that for γ-Fe₂O₃ (pH₀ 6.2), which was determined by the method of potentiometrical titration of oxide suspension in the nitrate solution. The introduction of surface-active ions Ba²⁺ and Cl^? changes the charge of passive iron surface: Ba²⁺ ions increase the electrode potential, while Cl^? ions decrease it. Comparing the pH₀ values for passive electrode and metal oxides, one can identify the composition of passive electrode surface.     

Comparison on effect of hydrophobicity on the antibacterial and antifungal activities of α-helical antimicrobial peptides

HPRP-A1, a 15-mer α-helical cationic peptide, was derived from N-terminus of ribosomal protein L1 (RpL1) of Helicobacter pylori. In this study, HPRP-A1 was used as a framework to obtain a series of peptide analogs with different hydrophobicity by single amino acid substitutions in the center of nonpolar face of the amphipathic helix in order to systematically study the effect of hydrophobicity on biological activities of -helical antimicrobial peptides. Hydrophobicity and net charge of peptides played key roles in the biological activities of these peptide analogs; HPRP-A1 and peptide analogs with relative higher hydrophobicity exerted broad spectrum antimicrobial activity against Gram-negative bacteria, Gram-positive bacteria and pathogenic fungi, but also showed stronger hemolytic activity; the change of hydrophobicity and net charge of peptides had similar effects with close trend and extent on antibacterial activities and antifungal activities. This indicated that there were certain correlations between the antibacterial mode of action and the antifungal mode of action of these peptides in this study. The peptides exhibited antimicrobial specificity for bacteria and fungi, which provided potentials to develop new antimicrobial drugs for clinical practices.     

Kinetic study of barrierless coagulation of sols in electrolyte solutions

The flow ultramicroscopy method is employed to investigate the coagulation kinetics of hydrosols of α-Fe₂O₃, α-Fe00H, γ-Al₂O₃, and Cr₂O₃ with electrolytes (NaCl and KCl) under conditions corresponding to the absence of an ion-electrostatic barrier. It is found that, at pH values corresponding to pH_IEP of particles, as well as at those differing from pH_IEP, but at sufficiently high ionic strengths of coagulating electrolytes, the observed rate of coagulation is close to or above that calculated in terms of the Smoluchowski theory of fast coagulation. The data obtained are analyzed and possible reasons for the observed effect are considered with regard to the fractal structure of aggregates, the polydispersity of initial particles, and the concepts of additional interparticle attractive forces arising at high electrolyte concentrations.     

Pairing Ion Effects in the Reversed Phase High Performance Liquid Chromatography of Peptides in the Presence of Alkylsulphonates

Abstract

The influence of pH and the concentration of the two lipophilic pairing ions, hexylsulphonate and camphor-10-sulphonate, on the retention of a group of small peptides to chemically bonded hydrocarbonaceous, microparticulate silicas has been further investigated. With low pH aqueous methanol mobile phases containing various concentrations of these surface active anions, the capacity factors of unprotected and C-protected peptides show similar dependencies on the concentration of the pairing ion. Column selectivity becomes essentially independent of pairing ion concentration above ca 25mM. At higher pH values, the influences of pairing ion interactions on peptide retention appear to diminish due to competing protic equilibria.     

Chromatofocusing of peptides on a sulfo-cation-exchange sorbent

Simple mobile phases containing no more than two active components were proposed for the formation of ascending pH gradients in a column filled with a sulfo-cation-exchange sorbent. The smoothest nearly linear pH gradients were obtained with the use of citric acid and Tris or NaH₂PO₄ and Tris as active components of the eluent and adjusting ionic strength (up to 0.1–0.3) in the starting solution or eluent; however, in the case of UV detection, the use of NaH₂PO₄ is preferable because of lower light absorption. Potentialities of the proposed approach in the chromatography of peptide mixtures on a sulfo-cation-exchange sorbent were demonstrated. Additions of acetonitrile to mobile phases improve the selectivity of the separation of peptides.