Prediction of peptide retention volumes in gradient reversed phase HPLC

A method of computation of retention volumes of linear peptides of known composition that contain no more than 25 amino acids in gradient reversed phase HPLC was developed. Tha method is suitable for various acetonitrile gradient profiles. The calculations were carried out on the basis of a statistical model, the parameters of which were experimental dependences of the retention of individual amino acids on acetonitrile concentration. The method developed was used to predict the chromatographic behavior of 34 peptides in four different acetonitrile gradients. The correlation coefficients between the predicted and experimental retention volumes were more than 0.9, and the average relative error of prediction was less than 15%.     

Prediction of retention volumes and UV spectra of peptides in reversed phase HPLC

A method for calculating retention volumes of linear peptides with known primary structures and the values of their UV absorption at chosen wavelengths in reversed phase HPLC are described. These parameters are calculated for every peptide on the basis of the contributions of its amino acid residues determining its degree of retention and its UV spectrum. The contribution values are experimentally found from chromatograms of the free amino acids obtained by multiwavelength photometric detection under the conditions of the peptide chromatography. Thirty peptides have been chromatographed for the evaluation of the proposed method, and the correlation coefficients between the calculated and the experimental retention volumes have been found to be 0.95.     

Prediction of retention volumes and UV spectra of peptides in reversed phase HPLC

A method for calculating retention volumes of linear peptides with known primary structures and the values of their UV absorption at chosen wavelengths in reversed phase HPLC are described. These parameters are calculated for every peptide on the basis of the contributions of its amino acid residues determining its degree of retention and its UV spectrum. The contribution values are experimentally found from chromatograms of the free amino acids obtained by multiwavelength photometric detection under the conditions of the peptide chromatography. Thirty peptides have been chromatographed for the evaluation of the proposed method, and the correlation coefficients between the calculated and the experimental retention volumes have been found to be 0.95.     

Determinations of catechols in small volumes of plasma using ion-pair reversed phase liquid chromatography/electrochemistry

A method with improved sensitivity for detection of catechols (CA) in small volumes of plasma using an ion-pair reversed phase HPLC system with electromechemical detection is presented. Fast isocratic separations were obtained by using 7.5 cm x 4.6 mm (i.d.) reversed phase columns with 3C18 3 micron silica particles. The CA:s L-DOPA, Noradrenaline (NA), Adrenaline (A), Dihydroxybenzylamine (DHBA, i.s.), DOPAC and Dopamine (DA) were separated in less than 4 min. The performance of three different electrochemical cells was compared with respect to hydrodynamic voltammogram, band broadening effect, linearity and detection limit. The sample preparation procedure using alumina extraction of CA:s, was modified to improve recoveries and decrease dilution factors. A modified carbon paste cell (CP-O) gave a response 4-8 times higher than what is previously reported for GC cells. Detection limits were: L-DOPA 80, NA 1.25, A 1.25, DHBA 0.4, DOPAC 1.25 and DA 0.6 pg/injection. Application to plasma from rat and fish (cod) under rest, exercise and stress is reported. The method allows determination of CA:s in small volumes of plasma (less than 500 microliter) obtained several times a day from the same animal even if it is small (less than 1/2 kg), is under rest and parts of the plasma sample are to be used for analysis of other parameters than CA:s.     

High performance liquid chromatography of peptide bioregulators, their fragments and derivatives. III. Regularities of sorption, prediction of retention and analysis of peptides by a reversed phase HPLC method

Parameters of statistical models of fully or partially protected peptides' retention on Zorbax ODS and Silasorb C18 have been compared. The proposed model can be used for non-protected linear and cyclic peptides. Special increments have to be introduced in calculation of hydrophobicity of these peptides.     

HPLC in reversed phase mode: Tool for investigation of kinetics of blackcurrant seed oil lipolysis in supercritical carbon dioxide

Blackcurrant (Ribes nigrum) seed oil is rich in alpha- and gamma-linolenic acids, the latter in particular being of potential use in medicine. The enzymatic hydrolysis of the oil was carried out in supercritical carbon dioxide using lipase Lipozyme as catalyst and changes in the composition of acylglycerols were recorded. Mono-, di-, and triacylglycerols and free fatty acids were separated by non-aqueous high-performance liquid chromatography in reversed phase mode and detected by UV diode array and 1H NMR detectors. Lipozyme was found to exert low specificity to individual fatty acids in the hydrolysed oil.     

HPLC enantiomeric resolution of novel aromatase inhibitors on cellulose- and amylose-based chiral stationary phases under reversed phase mode

The chiral resolution of seven aromatase inhibitors (four triazole derivatives (Ia, Ib, Ic, and Id) and three tetrazole derivatives (IIa, IIb, and IIc)) was achieved on Chiralcel OJ-R [cellulose tris (4-methyl benzoate)], Chiralcel OD-RH [cellulose tris (3,5-dimethylphenyl carbamate)], and Chiralpak AD-RH [amylose tris (3,5-dimethylphenyl carbamate)] chiral stationary phases. The mobile phases used were A: 2-PrOH-MeCN (90:10, v/v); B: 2-PrOH-MeCN (50:50, v/v); C: MeCN-H(2)O (50:50, v/v); D: MeCN-H(2)O (80:20, v/v); and E: MeCN-H(2)O (95:05, v/v). The flow rate was 0.5 mL/min for all the mobile phases. The resolution capability of these chiral stationary phases were in the order Chiralpak AD-RH > Chiralcel OD-RH > Chiralcel OJ-R. The values of alpha and Rs of the resolved enantiomers of the aromatase inhibitors varied from 1.02-5.63 and 1. 12-6.72, respectively.     

Quantitative determination of lysophosphatidic acid by LC/ESI/MS/MS employing a reversed phase HPLC column

Lysophosphatidic acid (LPA) is a class of lipids that play multiple biological functions. Several reports show that they are potential biomarkers for diagnosing ovarian cancer. Therefore, it is necessary to accurately quantify their levels in biological samples. Here we report a high throughput LC/ESI/MS/MS (liquid chromatography electrospray tandem mass spectrometry) method employing a reversed phase C18 column to quantify LPA. In this method, a [(13)C(16)] labeled 16:0 LPA is used as the internal standard and the lipids are extracted out from biological samples using Bligh-Dyer method under highly acidic condition. The total run time is 8min. The detection limits of the assay reach fmol level and the CV% of the assay are within 10%. Using this method, we quantify the levels of six LPA species (16:0, 18:2, 18:1, 18:0, 20:4, and 22:6 LPA) in plasma samples. We find that some unknown compounds present in plasma can interfere with the quantification of LPA if they are not well separated from LPA. These unknown compounds are more hydrophobic than LPA and can be removed by thin-layer chromatography (TLC). We also find that the levels of LPA species in human plasma generally follow the order: 18:2 LPA>16:0 LPA, 20:4 LPA>18:1 LPA, 22:6 LPA, and 18:0 LPA.     

Prediction of protein retention in hydrophobic interaction chromatography

Hydrophobic interaction chromatography (HIC) is a powerful technique for protein separation. This review examines methodologies for predicting protein retention time in HIC involving elution with salt gradients. The methodologies discussed consider three-dimensional structure data of the protein and its surface hydrophobicity. Despite their limitations, the methods discussed are useful in designing purification processes for proteins and easing the tedious experimental work that is currently required for developing purification protocols.     

Specific volumes of unsaturated phosphatidylcholines in the liquid crystalline lamellar phase

The specific volumes of seven 1,2-diacyl-sn-glycero-3-phosphocholines with symmetric, unbranched acyl chains containing one, four, or six cis double bonds per chain, or with a saturated sn-1 chain and one, four, or six cis double bonds in the sn-2 chain were determined by the neutral buoyancy method. Experiments were conducted in the liquid crystalline lamellar phase over the temperature range from 5 to 35 °C. It is demonstrated that the molecular volume of phosphatidylcholines can be well approximated as the sum of a constant volume of the polar lipid head region and the temperature-dependent volumes of hydrocarbon chain CH₂, CH, and terminal CH₃ groups. A linear dependence of chain segment volumes on temperature was observed. A self-consistent set of partially temperature-dependent volumes is obtained that allows prediction of phosphatidylcholine molecular volumes within very tight error margins.     

Specific volumes of unsaturated phosphatidylcholines in the liquid crystalline lamellar phase

The specific volumes of seven 1,2-diacyl-sn-glycero-3-phosphocholines with symmetric, unbranched acyl chains containing one, four, or six cis double bonds per chain, or with a saturated sn-1 chain and one, four, or six cis double bonds in the sn-2 chain were determined by the neutral buoyancy method. Experiments were conducted in the liquid crystalline lamellar phase over the temperature range from 5 to 35 degrees C. It is demonstrated that the molecular volume of phosphatidylcholines can be well approximated as the sum of a constant volume of the polar lipid head region and the temperature-dependent volumes of hydrocarbon chain CH2, CH, and terminal CH3 groups. A linear dependence of chain segment volumes on temperature was observed. A self-consistent set of partially temperature-dependent volumes is obtained that allows prediction of phosphatidylcholine molecular volumes within very tight error margins.     

Protein extration from an aqueous phase into a reversed micellar phase: Effect of water content and reversed micellar composition

In the system composed of the cationic surfactant TOMAC (10 mM), the nonionic (co)surfactant Rewopal HV5 (2 mM), and octanol (0.1% v/v) in isooctane, reversed micelles are formed upon contact with an aqueous phase containing 50 mM ethylene diamine. alpha-Amylase can be transferred from the aqueous phase into reversed micelles in the pH range 9.5 to 10.5 and re-extracted into a second aqueous phase of different composition. The size of the reversed micelles (as reflected in the water content of the organic phase) can be varied by changes in percentage of octanol, type of counterion in the aqueous phase, or in the number of ethoxylate head groups of the nonionic surfactant. An increase in size results in transfer at lower pH values. Experiments in which the charge density in the reversed micellar interface was changed by incorporation of charged derivatives of the nonionic surfactant, without influencing the water content, revealed that an increased charge density facilitated transfer, resulting in a broader transfer profile. Replacement of TOMAC by other quaternary ammonium surfactants differing in number and length of tails revealed that, of the 14 surfactants tested, only 2 gave appreciable amounts of transfer. The amount of transfer is related to the dynamics of phase separation of the surfactants: those giving a poor phase separation inactivate the enzyme. This inactivation is caused by electrostatic interactions between the charged surfactant head groups and charged groups on the enzyme. Electrostatic interactions are the first step of transfer, and can result in either incorporation in a reversed micelle, or, if reversed micelle formation is slow, in enzyme inactivation. (c) 1995 John Wiley & Sons, Inc.     

Aligning LC peaks by converting gradient retention times to retention index of peptides in proteomic experiments

MOTIVATION: Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a powerful tool in proteomics studies, but when peptide retention information is used for identification purposes, it remains challenging to compare multiple LC-MS/MS runs or to match observed and predicted retention times, because small changes of LC conditions unavoidably lead to variability in retention times. In addition, non-contiguous retention data obtained with different LC-MS instruments or in different laboratories must be aligned to confirm and utilize rapidly accumulating published proteomics data. RESULTS: We have developed a new alignment method for peptide retention times based on linear solvent strength (LSS) theory. We found that log k(0) (logarithm of retention factor for a given organic solvent) in the LSS theory can be utilized as a 'universal' retention index of peptides (RIP) that is independent of LC gradients, and depends solely on the constituents of the mobile phase and the stationary phases. We introduced a machine learning-based scheme to optimize the conversion function of gradient retention times (t(g)) to log k(0). Using the optimized function, t(g) values obtained with different LC-MS systems can be directly compared with each other on the RIP scale. In an examination of Arabidopsis proteomic data, the vast majority of retention time variability was removed, and five datasets obtained with various LC-MS systems were successfully aligned on the RIP scale.     

Informatics for peptide retention properties in proteomic LC-MS

Retention times in HPLC yield valuable information for the identification of various analytes and the prediction of peptide retention is useful for the identification of peptides/proteins in LC-MS-based proteomics. Informatics methods such as artificial neural networks and support vector machines capable of solving nonlinear problems made possible the accurate modeling of quantitative structure-retention relationships of peptides (including large polymers) up to 5 kDa to which classical linear models cannot be applied, as well as the proteome-wide prediction of peptide retention. Proteome-wide retention prediction and accurate mass-information facilitate the identification of peptides in complex proteomic samples. In this review, we address recent developments in solid informatics methods and their application to peptide-retention properties in 'bottom-up' shotgun proteomics. We also describe future prospects for the standardization and application of retention times.     

Prediction of formic acid chromatogram in gradient elution chromatography

Optimal operation in chromatography is needed to save operation time and the solvent used in multiple chromatographic runs. To this end, many simulation studies of chromatography process have been performed. The relationship between the distribution coefficient and the ionic strength is important in gradient elution ion chromatography. Experimental runs and computer simulation were carried out under linear gradient elution conditions in order to compare the experiments and the simulation. Experiments were performed with formic acid under isocratic conditions to determine the simulation equation parameters. Computer simulation was based on three equations which related distribution with ionic strength as follows;K=αI ^−β, K=A+BI+Cl² and. The effects of gradient slope on the chromatograms are discussed, and good agreement between the experimental and the simulated results is shown.     

Nuclear magnetic resonance evidence for retention of a lamellar membrane phase with curvature in the presence of large quantities of the HIV fusion peptide

The HIV fusion peptide (HFP) is a biologically relevant model system to understand virus/host cell fusion. ²H and ³¹P NMR spectroscopies were applied to probe the structure and motion of membranes with bound HFP and with a lipid headgroup and cholesterol composition comparable to that of membranes of host cells of HIV. The lamellar phase was retained for a variety of highly fusogenic HFP constructs as well as a non-fusogenic HFP construct and for the influenza virus fusion peptide. The lamellar phase is therefore a reasonable structure for modeling the location of HFP in lipid/cholesterol dispersions. Relative to no HFP, membrane dispersions with HFP had faster ³¹P transverse relaxation and faster transverse relaxation of acyl chain ²H nuclei closest to the lipid headgroups. Relative to no HFP, mechanically aligned membrane samples with HFP had broader ³¹P signals with a larger fraction of unoriented membrane. The relaxation and aligned sample data are consistent with bilayer curvature induced by the HFP which may be related to its fusion catalytic function. In some contrast to the subtle effects of HFP on a host-cell-like membrane composition, an isotropic phase was observed in dispersions rich in phosphatidylethanolamine lipids and with bound HFP.     

Ionic partial molar volumes of density gradient salts at finite concentrations.

A general method is presented for estimating concentration-dependent partial molar volumes for individual ions of heavy density gradient salts used in the isopycnic ultracentrifugation of charged macromolecules.