Use Of Reversed-phase Chromatography Research Articles

Acetonitrile adduct analysis of underivatised amino acids offers improved sensitivity for hydrophilic interaction liquid chromatography tandem mass-spectrometry

LC-MS/MS method development for native amino acid detection can be problematic due to low ionisation efficiencies, in source fragmentation, potential for cluster ion formation and incorrect application of chromatography techniques. This has led to the majority of the scientific community derivatising amino acids for more sensitive analysis. Derivatisation has several benefits including reduced signal-to-noise ratios, more efficient ionisation, and a change in polarity, allowing the use of reverse phase chromatography. However, derivatisation of amino acids can be expensive, requires additional sample preparation steps, is more time consuming and increases sample instability, due to the most derivatised amino acids only be stable for finite amount of time. While showing initial promise, development of reliable hydrophilic interaction liquid chromatography (HILIC) separation methods has presented difficulties for the analyst including irreproducible separation and poor sensitivity. This study aimed to find a means to improve the detection sensitivity of the 20 protein amino acids by HILIC-MS/MS. We describe the use of previously undescribed amino acid-acetonitrile (ACN) adducts to improve detection of 16 out of the 20 amino acids. While all amino acids examined did form an ACN adduct, 4 had low intensity adduct formation compared to their protonated state, 3 of which are classified as basic amino acids. For 15 of the 20 amino acids tested, we used the ACN adduct for both quantification and qualification ions and demonstrated a significant enhancement in signal-to-noise ratio, ranging from 23 to 1762% improvement. Lower LODs, LOQs and lower ranges of linearity were also achieved for these amino acids. The optimised method was applied to a human neuroblastoma cell line (SH-SY5Y) with the potential to be applied to other complex sample types. The improved sensitivity this method offers simplifies sample preparation and reduces the costs of amino acid analysis compared to those methods that rely on derivatisation for sensitivity.

Efficient expression and isolation of recombinant human interleukin-11 (rhIL-11) in Pichia pastoris

Current source of recombinant human interleukin-11 (rhIL-11) is isolated from a fusion protein expressed by E. coli that requires additional enterokinase to remove linked protein, resulting in product heterogeneity of N-terminal sequence. Due to lack of glycosylation, rhIL-11 is suitable to be expressed by yeast cells. However, the only available yeast-derived rhIL-11 presents an obstacle in low production yield, as well as an unamiable process, such as the use of reverse-phase chromatography employing plenty of toxic organic solvents. Our findings showed that the low yield was due to self-aggregation of rhIL-11. A novel process recovering bioactive rhIL-11 from the yeast secretory medium therefore has been developed and demonstrated, involving fermentation from Pichia pastoris, followed by a two-phase extraction to precipitate rhIL-11. After renaturing, the protein of interest was purified by a two-column step, comprising a cation-exchanger, and a hydrophobic interaction chromatography in tandem at high sample loads that was facile and cost-effective in future scale-up. Identity and quality assessments confirmed the expected amino acid sequence without N-terminal heterogeneity, as well as high quality in potency and purity. Such a process provides an alternative and adequate supply of the starting material for the PEGylated rhIL-11.

Scalable Synthesis of a Nucleoside Phosphoramidate Prodrug Inhibitor of HCV NS5B RdRp: Challenges in the Production of a Diastereomeric Mixture

A scalable process is described for the synthesis of 2′-C-methylguanosine-5′-[2-[(3-hydroxy-2,2-dimethyl-1-oxopropyl)thio]ethyl-N-benzylphosphoramidate], a nucleotide prodrug inhibitor of hepatitis C virus NS5B polymerase. The route features the use of phenylboronic acid to transiently protect the 2′,3′-hydroxyls of 2′-C-methylguanosine under mild conditions. The requirement to produce a 1:1 phosphorus diastereomeric mixture precluded the use of traditional crystallization techniques. High sensitivity of the drug substance to acidic and basic conditions and its preferred solubility in mixed aqueous–organic solvents presented additional processing challenges. The use of reverse phase chromatography for the final purification was eliminated by the development of a dual liquid–liquid extraction protocol, which removed both non-polar and polar impurities whilst maintaining the 1:1 diastereomeric ratio. Ethylene sulfide, a potential genotoxic impurity that was observed at significant levels in the original pro...

Separation of PEGylated variants of ribonuclease A and apo-α-lactalbumin via reversed phase chromatography

The covalent attachment of polyethylene glycol (PEG) molecules to pharmaceutical proteins, “PEGylation”, often results in a population of conjugate species that includes differing numbers and locations of attached PEG chains. As some portion of this population may be biologically inactive, a challenging separation problem arises. An interesting alternative to the size-based resolution of these conjugates involves the use of reversed phase chromatography (RPC), treating the PEG moieties as hydrophobic purification tags. The use of RPC raises concerns about protein denaturation in the mobile and on the stationary phase. Here, the potential dual role of conjugated PEG chains as both group-specific separation tags and as steric or structural stabilizers in RPC was explored. In this work, RPC with C18-based media was used to resolve PEGylation number variants of ribonuclease A (RNase A) and apo-α-lactalbumin (apo-αLac) in a neutral pH mobile phase. While the attachment of 20kDa PEG molecules did not modify the structures of RNase A and apo-αLac, as confirmed by structural analysis using circular dichroism, exposure to the mobile phase modifier, acetonitrile, and to the C18 media during separation resulted in perturbations to both the secondary and tertiary structures of all species studied. RNase A experienced small perturbations that were mediated to some extent by PEGylation; these results were consistent with activity assays which showed that PEGylated RNase A species retained native-like activity after RPC separation. Apo-αLac, a more hydrophobic and less stable protein than RNase A, experienced extensive structural perturbations regardless of PEGylation state. The temperature of the mobile phase was found to strongly influence chromatographic separation of PEG-conjugates with conjugate species becoming more strongly retained with increasing temperature. This work shows that it is feasible to employ RPC with neutral pH mobile phases to resolve PEG conjugate number heterogeneity.

Characterization, Localization, and Stage-Dependent Gene Expression of Gonadotropin Receptors in Chub Mackerel (Scomber japonicus) Ovarian Follicles

The pituitary gonadotropins (GtHs), follicle-stimulating hormone (FSH) and luteinizing hormone (LH), are key regulators of gametogenesis in teleosts. However, little is known about the physiological mechanisms by which GtHs regulate asynchronous oocyte development in multiple-spawning marine fishes. We cloned cDNAs encoding GtH receptors (FSHR and LHR) from chub mackerel (Scomber japonicus). FSH and LH were purified by anion-exchange chromatography, gel filtration, and concanavalinA-agarose. When expressed in mammalian cells, FSHR and LHR responded strongly to their own ligands. By separating LH into two subunits by the use of reverse-phase chromatography, we found that the beta-subunit is responsible for signal transduction and the alpha-subunit may be important for holding hormone-receptor complex. In situ hybridization showed that only fshr was expressed in prefollicle and granulosa cells in oocytes at the perinucleolus and cortical alveolus stages, suggesting that FSH is involved in the primary and early secondary growth of oocytes. In ovarian follicles during vitellogenesis, both fshr and lhr were expressed in granulosa and thecal cells, and lhr was strongly expressed during germinal vesicle migration (GVM). Real-time PCR analysis of stage-dependent fshr and lhr expression showed that fshr expression was high in ovarian follicles throughout vitellogenesis and decreased during GVM, whereas lhr expression was low in early vitellogenesis, but increased markedly in the late phase of vitellogenesis, remaining high during GVM. These findings suggest that switching of the expression of FSHR to LHR controls the effects of FSH and/or LH on vitellogenesis and final oocyte maturation via steroid production in granulosa and thecal cells.

Ganoderma species discrimination by dual-mode chromatographic fingerprinting: A study on stationary phase effects in hydrophilic interaction chromatography and reduction of sample misclassification rate by additional use of reversed-phase chromatography

Acetonitrile-water extracts of several Ganoderma species - a mushroom being used in Traditional Chinese Medicine - were analysed by liquid chromatography-UV detection in hydrophilic interaction chromatography (HILIC) and reversed-phase (RP) elution modes. A set of six polar stationary phases was used for HILIC runs. These columns had remarkably different separation properties under binary gradient conditions as evinced by hierarchical cluster analysis on retention patterns of seven test compounds. Complementary measurements of RP chromatograms were carried out on a C(18) packing. Injection precision (n=5) and intra-day precision (n=5) were each <2.0% RSD (HILIC) and <0.7% RSD (RP) for relative retention times of main characteristic peaks of a sample extract while for relative peak areas RSD values were max. 6.8%. Repetitive analysis (n=7) of a processed sample stored in the autosampler tray for 48h was used to confirm within-sequence sample stability. Eleven Ganoderma lucidum samples served as training set for the construction of column-specific simulated mean chromatograms. Validation with twelve samples comprising G. lucidum, Ganoderma sinense, Ganoderma atrum, and Ganoderma tsugae by correlation coefficient based similarity evaluation of peak patterns showed that a discrimination of G. lucidum from other Ganoderma species by means of chromatographic fingerprints is conceptually possible on all columns, except of a bare silica packing. The importance of the combined use of RP and HILIC fingerprints to improve the rate of correct sample classification was demonstrated by the fact that each one G. sinense specimen was wrongly assigned being G. lucidum by all HILIC fingerprints but not the RP fingerprint and vice versa. The present data revealed that (i) the analysis of complex biological materials by quasi orthogonal chromatographic modes such as HILIC and RP may deliver more discriminative information than single-mode approaches which strengthens the reliability of fingerprint-based sample classification and (ii) different retention and selectivity characteristics of polar bonded silica packings in the HILIC elution mode may only have a minor impact on chemometric sample discrimination capabilities in such kind of pattern-oriented metabolomics separation problems.

Ultra‐fast tandem mass spectrometry scanning combined with monolithic column liquid chromatography increases throughput in proteomic analysis

Liquid chromatography combined with electrospray ionization mass spectrometry (LC/ESI-MS) has been used successfully for the characterization of biomolecules in proteomics in the last few years. This methodology relied largely on the use of reversed-phase chromatography, in particular C18-based resins, which are suitable for separation of peptides. Here we show that polymeric [polystyrene divinylbenzene] monolithic columns can be used to separate peptide mixtures faster and at a higher resolution. For 500 fmol bovine serum albumin, up to 68% sequence coverage and Mascot Mowse scores of >2000 were obtained using a 9 min gradient on a monolithic column coupled to an ion trap mass spectrometer with ultra-fast MS/MS scan rates. In order to achieve similar results using C18 columns, it was necessary to extend gradient times to 30 min. In addition, we demonstrate the utility of this approach for the analysis of whole Escherichia coli cell lysates by one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (1D-SDS-PAGE) in combination with LC/MS/MS using 4 min gradients on monolithic columns. Our results indicate higher throughput capabilities of monolithic columns (3-fold gain in time or more) for conventional proteomics applications, such as protein identification and high sequence coverage usually required for detection of post-translational modifications (PTMs). Further optimization of sensitivity and quality of sequence information is discussed, in particular when combined with mass spectrometers that have very fast MS-MS/MS switching and scanning capabilities.

Reversed-phase ion-pairing liquid chromatography/ion trap mass spectrometry for the analysis of negatively charged, derivatized glycans.

The significant complexity, similar polarity and lack of ionizable sites make the analysis of glycans an analytical challenge. These compounds are often derivatized and separated by normal-phase high-performance liquid chromatography (HPLC) or capillary electrophoresis (CE) followed by UV or fluorescence detection. Due to widespread use of reversed-phase chromatography coupled to electrospray mass spectrometry as an analytical tool, our laboratory has developed this methodology for the analysis of glycans derivatized with a negatively charged tag, 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS). It is possible to exploit the ability of this negatively charged tag to interact with a mobile phase ion-pairing reagent, allowing retention on a reversed-phase C(18) column for subsequent on-line UV or MS analysis. ANTS-derivatized samples, including a maltooligosaccharide ladder and glycans released from bovine ribonuclease B, bovine fetuin, and chicken ovalbumin, were analyzed using this method. In addition to reversed-phase retention, ribonuclease B and ovalbumin derivatives displayed highly desirable isomeric separation. With the use of mass spectrometric detection for glycan identity, this allowed relative quantitation of individual components.

Increased cleavage of the c-terminal serine from alpha-A crystallin present in the high molecular weight aggregate fraction from human and bovine lenses

PURPOSE. To determine the relative amount of serine-173 cleavage from the C-terminus of alpha-A crystallin from the high molecular weight aggregate fraction and lower molecular weight alpha crystallin fraction from water soluble proteins of human and bovine lenses. METHODS. Using gel permeation chromatography, the high molecular weight aggregate fraction and lower molecular weight alpha crystallin fractions were purified from soluble proteins of human lenses and mature bovine lenses. Alpha-A crystallin present in these fractions was digested with trypsin, followed by the use of reverse phase chromatography and synthetic peptide standards to identify and quantitate the relative amount of in vivo cleavage of C-terminal serine –173. RESULTS. For all human and bovine lenses studied, alpha-A crystallin in the high molecular weight aggregate fraction had undergone more truncation at the C-terminus, than the alpha-A crystallin present in the lower molecular weight alpha crystallin fraction from the same lens. CONCLUSIONS. Relative to the alpha-A crystallin present in the lower molecular weight alpha fraction, the alpha-A crystallin present in the high molecular weight aggregate fraction from the same human and bovine lenses has undergone increased cleavage of the C-terminal serine residue, suggesting a common mechanism that may contribute towards formation of protein aggregation in vivo.

19] Analysis of synthetic peptides by high-performance liquid chromatography

High-performance liquid chromatography (HPLC) has proved extremely versatile for the isolation/purification of peptides varying widely in their sources, quantity, and complexity. High-performance chromatographic techniques are suited to the purification of a single peptide from the kind of complex peptide mixtures encountered following solid-phase peptide synthesis, where impurities are closely related to the peptide of interest (deletion, terminated, or chemically modified peptides), perhaps missing only one amino acid residue, and, hence, may be difficult to separate. This chapter focuses on HPLC applications of particular interest to researchers utilizing solid-phase synthesis methods: (1) problem-solving approaches to difficult peptide separations, (2) HPLC monitoring of formation of desired product, (3) and HPLC as a diagnostic tool to detect unwanted side-chain modification. The use of reversed-phase chromatography (RP-HPLC), the most important mode of HPLC for synthetic peptidepurification, is focussed; in addition, the exciting potential of a novel mixed-mode HPLC technique, hydrophilic interaction/cation-exchange chromatography (HILIC/CEC), is demonstrated.

Insect neuropeptide F (NPF)-related peptides: Isolation from colorado potato beetle (Leptinotarsa decemlineata) brain

Two novel neuropeptides with neuropeptide F (NPF)-like immunoreactivity have been isolated from brain extracts of the Colorado potato beetle. Purification was achieved primarily by use of reverse phase chromatography including initial C-18 Sep-Pak cartridges and 4 subsequent analytical HPLC columns. Combined data from automated Edman degradation, immunochemical analysis, u.v. absorbance and mass spectrometry led to the elucidation of their full primary structures. The deduced sequences are: Ala-Arg-Gly-Pro-Gln-Leu-Arg-Leu-Arg-Phe-NH 2 (ARGPQLRLRFamide) and Ala-Pro-Ser-Leu-Arg-Leu-Arg-Phe-NH 2 (APSLRLRFamide). On the basis of their primary structure both peptides can be appended to the invertebrate group of neuropeptide Y (NPY)-like peptides, generally referred to as NPFs. We suggest these peptides to be designated Led-NPF-1 and Led-NPF-2.

High-level synthesis in Escherichia coli of shortened and full-length human acidic fibroblast growth factor and purification in a form stable in aqueous solutions

A highly efficient expression for human acidic fibroblasts growth factor (aFGF) has been assembled to direct the synthesis of both shortened and native full-length aFGF. The full-length aFGF-154 form of the protein had not been produced before in Escherichia coli by genetic engineering, and is obtained with its initiator methionine removed. The high production of the aFGF allows one to circumvent the use of reversed-phase chromatography (RPC) during the purification procedure. Here, it is shown that RPC, routinely used to obtain pure preparations of recombinant aFGF, modifies its chemical and physical properties in an unfavorable manner.

Experiments on 14C Dating of Contaminated Bone Using Peptides Resulting from Enzymatic Cleavage of Collagen

We describe the use of collagenase to digest ancient collagen samples to obtain a peptide mixture, and the use of reversed-phase chromatography to purify these peptides. The isolation of the tripeptides glycine-proline-hydroxyproline and glycine-proline-alanine is especially encouraging.

Use of reverse-phase chromatography in the Maxam-Gilbert method of DNA sequencing A step toward automation

Reverse-phase chromatography has been used for carrying out Maxam-Gilbert sequencing reactions. The DNA loss has been minimized. Time-consuming ethanol precipitation and lyophilization of piperidine have been eliminated. The method has been used successfully in sequencing oligonucleotides as well as large DNA fragments. Based upon these modifications, we have developed a semiautomatic device for carrying out the Maxam-Gilber sequencing reactions. It is also possible to fully automate the Maxam-Gilbert method of DNA sequencing, and we present the diagram of a fully automated set up. The development of such a machine should expedite the sequencing of the large number of genes being cloned.

Use of reversed-phase chromatography in carbohydrate analysis

High-performance liquid chromatography of carbohydrates on different types of reversed-phase column with refractive index detection and water as the el It was concluded that decreasing the column temperature results in increased retention times and better resolution; decreasing the length of the alkyl