Application of reversed-phase high-performance liquid chromatography to the separation of peptides from phosphorylated and dephosphorylated casein hydrolysates (original) (raw)
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Journal of Chromatography A, 2005
Bioactive peptides and tryptic digests of various proteins were separated under acidic and alkaline conditions by ion-pair-reversed-phase high-performance liquid chromatography (RP-HPIPC) in 200 m I.D. monolithic, poly(styrene-divinylbenzene)-based capillary columns using gradients of acetonitrile in 0.050% aqueous trifluoroacetic acid, pH 2.1, or 1.0% triethylamine-acetic acid, pH 10.6. Chromatographic performances with mobile phases of low and high-pH were practically equivalent and facilitated the separation of more than 50 tryptic peptides of bovine serum albumin within 15-20 min with peak widths at half height between 4 and 10 s. Neither a significant change in retentivity nor efficiency of the monolithic column was observed during 17-day operation at pH 10.6 and 50 • C. Upon separation by RP-HPIPC at high-pH, peptide detectabilities in full-scan negative-ion electrospray ionization mass spectrometry (negESI-MS) were about two to three times lower as compared to RP-HPIPC at low-pH with posESI-MS detection. Tandem mass spectra obtained by fragmentation of deprotonated peptide ions in negative ion mode yielded interpretable sequence information only in a few cases of relatively short peptides. However, in order to obtain sequence information for peptides separated with alkaline mobile phases, tandem mass spectrometry (MS/MS) could be performed in positive ion mode. The chromatographic selectivities were significantly different in separations performed with acidic and alkaline eluents, which facilitated the fractionation of a complex peptide mixture obtained by the tryptic digestion of 10 proteins utilizing off-line, two-dimensional RP-HPIPC at high pH × RP-HPIPC at low pH and subsequent on-line identification by posESI-MS/MS.
Journal of Chromatography A, 2009
This review discusses different liquid chromatographic and capillary electrochromatographic approaches to the separation and quantitation of peptides using silica-based and polymeric-based columns with emphasis on liquid chromatography. Mass spectrometry detection and quantitation of peptides using labeled and label-free procedures, will also be discussed, as well as the effect of amino acids' properties on the solubility of peptides, an important parameter that influences the selection of the mobile phase. A discussion of different column packing materials, reversed-phase, cyclodextrins, macrocyclic antibiotics, porous graphitic carbon, mixed-phases, and normal-phase will be included, as well as a short discussion of multi-dimensional approaches for the separation of complex peptide mixtures.
Separation of peptides by strong cation-exchange high-performance liquid chromatography
Journal of Chromatography A, 1985
The effects of pH and gradient conditions on the separation of a series of ten peptides (9-36 residues) and carboxamidomethylated troponin I (CM-TnI, 178 residues) on a new commercially available strong cation-exchange silica based 300-A column (Synchropak S300) were examined. The elution times of the peptides were linear with respect to their net charge at pH 3.0 and pH 6.5. The basic protein CM-TnI (pZ % 9.5) and peptides with net charges from + 2 to + 10 were separated with linear AB salt gradients varying from 5 mM to 10 mM B per min (A = 5 mM KH2P04 buffer, pH 6.5 or 3.0; B = 5 mM KH2P04 buffer, pH 6.5 or 3.0, containing 1 M KCl). All peptides and CM-TnI were eluted with KC1 concentrations below cu. 0.6 M. The advantage of performing cation-exchange chromatography over anionexchange chromatography was demonstrated for the separation of peptides which, while acidic or weakly basic at neutral pH, through protonation of the acidic functions results in positively charged peptides at pH 3.0. 0021-9673/85/%03.30 0 1985 Elsevier Science Publishers B.V.
Journal of Chromatography A, 1983
Weak anion-exchange and reversed-phase high-performance liquid chromatographic methods for peptide separations were compared using a tryptic digest of "rat small myelin basic protein". In these experiments, a number of tryptic peptides that were-not resolved on the reversed-phase column could be separated on the weak anion-exchange column, and in other instances, as might be expected, reversed-phase chromatography provided better resolution of certain peptides than did the weak anion-exchange method. The results obtained strongly suggest that the combined use of these two methods of separation, which utilize different selectivities, can provide an excellent improvement in resolving power for a number of peptide separations.
HPLC Analysis and Purification of Peptides
Methods in Molecular Biologytm
High-performance liquid chromatography (HPLC) has proved extremely versatile over the past 25 yr for the isolation and purification of peptides varying widely in their sources, quantity and complexity. This article covers the major modes of HPLC utilized for peptides (size-exclusion, ion-exchange, and reversed-phase), as well as demonstrating the potential of a novel mixed-mode hydrophilic interaction/cation-exchange approach developed in this laboratory. In addition to the value of these HPLC modes for peptide separations, the value of various HPLC techniques for structural characterization of peptides and proteins will be addressed, e.g., assessment of oligomerization state of peptides/proteins by sizeexclusion chromatography and monitoring the hydrophilicity/hydrophobicity of amphipathic-helical peptides, a vital precursor for the development of novel antimicrobial peptides. The value of capillary electrophoresis for peptide separations is also demonstrated. Preparative reversed-phase chromatography purification protocols for sample loads of up to 200 mg on analytical columns and instrumentation are introduced for both peptides and recombinant proteins.
Food Chemistry, 2014
Numerous peptides generated by triptic hydrolysis of micellar casein and β-casein from bovine milk have been reported to exhibit bio-functional or techno-functional activities. In this study, the focus was placed on the quantification of eleven potential functional peptides generated by enzymatic hydrolysis with trypsin. The identity of the target peptides was verified by LC-ESI-MS/MS and a reverse phase high performance liquid chromatography method (RP-HPLC) was established to quantify the peptides. The quantification was based on calibration curves drawn using synthesised purified peptides. The regression analysis comparing >30 standard measurements of each peptide standard showed a regression coefficient R(2)>0.9958. The inter-day repeatability of the quantification method was always within a relative error of 0.2-6.5%, while the relative error of the accuracy for reproducibility was in the range of 0.1-2.4%. The established method was successfully applied for the quantitative analysis of the eleven functional peptides in different dairy fractions generated by cross-flow ultrafiltration.
Downstream Processing of Therapeutic Peptides by Means of Preparative Liquid Chromatography
Molecules
The market of biomolecules with therapeutic scopes, including peptides, is continuously expanding. The interest towards this class of pharmaceuticals is stimulated by the broad range of bioactivities that peptides can trigger in the human body. The main production methods to obtain peptides are enzymatic hydrolysis, microbial fermentation, recombinant approach and, especially, chemical synthesis. None of these methods, however, produce exclusively the target product. Other species represent impurities that, for safety and pharmaceutical quality reasons, must be removed. The remarkable production volumes of peptide mixtures have generated a strong interest towards the purification procedures, particularly due to their relevant impact on the manufacturing costs. The purification method of choice is mainly preparative liquid chromatography, because of its flexibility, which allows one to choose case-by-case the experimental conditions that most suitably fit that particular purification...
Journal of Chromatography A, 1988
Hydrophobic interaction chromatography (HIC) was examined as an alternative to reversed-phase chromatography (RPC) for peptide separations by highperformance liquid chromatography. With small peptides, selectivity was similar in both modes. This was the case with commercially available standards and with a set of synthetic peptides having the same amino acid composition but different sequences. Column efficiency was higher in RPC. HIC possesses several other disadvantages, including significant baseline changes during gradient elution and a requirement for non-volatile mobile phases, which complicates peptide isolation. Thus, RPC is still the method of choice for most small peptides. Marked differences in selectivity were noted with small proteins and polypeptides large enough to possess tertiary structure. Good results were also obtained by HIC in the case of some peptides that could not be purified at all by RPC, due to aggregation or poor binding or recovery. Thus, in these cases, HIC is a useful alternative to RPC for peptide purification.
Journal of Chromatography B: Biomedical Sciences and Applications, 1996
HPLC and CE have been applied to the separation of some newly synthesized substances, including nonapeptides from the intrachinary region of insulin, insulin-like growth factors I and II (IGF I and II) and some penta-and hexapeptides. All the peptides are satisfactorily separated using a reversed-phase HPLC system with a C~8 stationary phase and mobile phases of 20-40% acetonitrile (v/v) and 0.2% trifluoroacetic acid in water (v/v). The best CE separation of IGF I and II has been achieved in a 30 mM phosphate buffer (pH 4-5), whereas 150 mM phosphoric acid (pH 1.8) is optimal for the insulin nonapeptides. The latter electrolyte is also suitable for the CE separation of the hexapeptides, as is a micellar system containing 20 mM borate-50 mM sodium dodecyl sulfate (pH 9.0). Complete CE resolution of the D-and L-forms is possible in a 50 mM phosphate buffer (pH 2.5) containing 10 mM fl-cyclodextrin. UV spectrophotometric detection was used throughout, at wavelengths from 190 to 215 nm. The CE procedures are, in general, preferable to HPLC separations, as they exhibit better separation efficiencies, are faster and consume smaller amounts of analytes and reagents.
High-performance size-exclusion liquid chromatography of protected peptides
Journal of Chromatography A, 1981
Methods for the analytical and preparative size-exclusion high-performance liquid chromatography of protected hydrophobic peptides in organic solvents were developed. Columns containing Sephadex LH-20 and various silica gels were used and the selectivity of each sorbent and the behaviour of the peptides on them were examined_ The effects of sample volume and concentration and the injection technique were elucidated in preparative-scale work. The applicability of various detection methods for identifying peptides is considered. INTRODUCT ION A separation method suitable for a wide variety of molecules, especially biopolymers, based on the difference in their molecular masses was proposed by Porath and Flodin over 20 years ago'. Over the past 4-5 years the traditional method of gel filtration on strongly swelling soft gels'* has developed into high-performance sizeexclusion liquid chromatography (SEHPLC). A number of factors contributed to this development, including the elaboration of techniques for column packing with sorbents of small particle size (5-10 m), and the application of semi-rigid and rigid sorbents, permitting a large increase in the rate and efficiency of the separation. Sizeexclusion chromatography involves very simple procedures and mild conditions, suitable even for such labile compounds as proteins and peptides. This work was devoted to the development of SEHPLC methods for protected peptides obtained during the synthesis of large peptides and proteins. As such peptides are usually insoluble in water or aqueous-organic solvent systems, it is impossible to use common methods for the analysis and purification of proteins and unprotected peptides such as ion-exchanges, reversed-phase6*7* and partition'*" chromatography or counter-current distribution. The limited solubility of long-chain protected peptides in non-polar media and the risk-of irreversible sorption restrict the application of adsorption chromatography, although there are some examples of the * The work of Bakkum et a[.' is an exception; the solubility of protected secretin and its fragments allowed reversed-phase chromatography to be used.