Purification and partial characterization of phospholipase A2 isoforms from human placenta.

Five isoforms of the human placental phospholipase A2 were identified and purified to near homogeneity. The purification of these enzymes involved gel permeation, ion-exchange and affinity chromatography. The apparent relative molecular mass of these proteins is 70,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These enzymes have pH optima of 7 and 8. Two-dimensional gel electrophoresis of these enzymes revealed distinct pH optima for each of the isoforms with values ranging from 4.0 to 6.5. Three of the isoforms require calcium for activity whereas the other two forms exhibit 50% of their maximum activity without the presence of calcium.     

Recombinant autofluorescent landmarks for standardization of electrophoretic migration of proteins

Unequivocal identification of unknown protein spot patterns in two-dimensional (2-D) electrophoresis still represents a major problem when performing comparative studies of different 2-D electrophoresis gels. Inhomogeneity of gels due to variations in the gel casting procedure, electroendoosmosis and heterogeneity of proteins are major contributions to variations in migration patterns. By fusing green fluorescent protein to a number of well-defined selected proteins (human lysozyme, initiation factor 5a (EIF5a), rapamycin-selective 25 kDa immunophilin (FKBP25), and heat shock protein 90 beta (hsp90)), the isoelectric points and the molecular mass were designed. Proteins were additionally tagged with the FLAG tag enabling rapid purification by immunoaffinity chromatography. The fusion proteins were expressed intracellularly in yeast to avoid heterogeneity caused by post-translational modifications. The quality and applicability was tested in 1-D and 2-D electrophoresis. Sharp bands or symmetric spots were obtained. The proteins are considered as a new generation of reference proteins for electrokinetic separation methods.     

Amino acid sequences of 60B8 antigens induced in HL-60 cells by 1,25-dihydroxyvitamin D3. The antigens are identical with macrophage-related protein-14 and -8

A differentiation antigen 60B8 appeared in human promyelocytic leukemia HL-60 cells which had been induced to differentiate into macrophage-like cells by treatment with 1,25-dihydroxyvitamin D3. The antigen was purified by immunoaffinity chromatography and separated into two proteins, 60B8-A and -B antigens, by reverse-phase high performance liquid chromatography (HPLC). Both proteins were digested with proteases, and the resulting peptides were subjected to amino acid sequence analysis after purification by reverse-phase HPLC. The amino acid sequences of 60B8-A and -B antigens were identical with those of the proteins MRP-14 and -8, respectively, which were recently predicted from the nucleotide sequences of their complementary deoxyribonucleic acid (cDNA) clones by Odink et al. (Nature (London), 330, 80 (1987)). Although they did not characterize the chemical properties of the two proteins, our results clearly indicate that macrophage-related protein (MRP)-14 and -8 are expressed without post-translational modification, except that the amino-terminus of MRP-14 is blocked, in differentiated HL-60 cells.     

Purification of ascitic fluid-derived murine monoclonal antibodies by anion-exchange and size-exclusion high-performance liquid chromatography

Ascitic fluid-derived murine monoclonal antibodies (MoAbs) of immunoglobulin (Ig) M and IgG isotypes (IgG1 and IgG2a subisotypes) were previously prepared against an isolate of Actinobacillus sp (CAs8C) for the purpose of identifying and characterizing outer membrane antigens on this bacterium. An attempt was made to purify these MoAbs by anion-exchange and size exclusion high-performance liquid chromatography (HPLC). Hybridomas producing the IgG1 and IgG2a MoAbs posed unique difficulties in that they also secreted irrelevant IgG2b MoAbs that were present in the ascitic fluids. Anion-exchange chromatography (Protein-Pak DEAE-5PW column), with a simultaneous change in gradients of pH and ionic strength, was used to purify IgG and as a first step in the purification of IgM. There was good separation of IgG2b from IgG2a, but not from IgG1. Size-exclusion chromatography (Protein-Pak 300 SW column) was required to complete the purification of IgM. The presence of MoAbs in the HPLC fractions was confirmed by discontinuous gradient polyacrylamide gel electrophoresis (denatured and either reduced or non-reduced conditions) and the enzyme-linked immunosorbent assay. HPLC-purified MoAbs were free from transferrin and albumin and retained their specificity for As8C.     

Affinity purification of Schistosoma japonicum glutathione-S-transferase and its site-directed mutants with glutathione affinity chromatography and immobilized metal affinity chromatography.

A C-terminally polyhistidine-tagged protein of Schistosoma japonicum glutathione-S-transferase, named as SjGST/His, and its Cys85-->Ser, Cys138-->Ser, and Cys178-->Ser site-directed mutants were prepared and highly expressed in Escherichia coli. Both immobilized metal affinity chromatography (IMAC) and glutathione (GSH) affinity chromatography were used to purify these four enzymes. All of them were purified with equal efficiency by Ni2+-chelated nitrilotriacetic acid agarose gel, but not by GSH Sepharose 4B gel. The protein amounts of wild-type and Cys85-->Ser enzymes purified by the latter gel were three to seven-fold greater than those of the other two enzymes purified by the same gel, while their specific activities were two-fold lower, presumably because of the occurrence of noncovalent aggregation. Both purification methods yielded highly pure enzymes, while there were minor amounts of inter- and intra-disulfide forms in the IMAC purified enzymes except for the Cys85-->Ser mutant. Addition of dithiothreitol to GSH-affinity purified enzymes shifted all of their mass spectra of matrix-assisted laser desorption/ionization-time of flight mass spectrometry toward low molecular-mass regions, while addition of GSH to IMAC purified enzymes shifted the spectra toward high molecular-mass regions. The shift values of wild-type enzyme were larger than those of the three mutants, indicating that the Cys85, Cys138, and Cys178 residues were S-thiolated by GSH during the GSH-affinity purification. This result was confirmed by isoelectric focusing. These findings suggest that IMAC is more efficient than the conventional GSH-affinity system for the purification of SjGST/His enzyme, especially for its mutants and fusion proteins.     

Quantification of chitinase and thaumatin-like proteins in grape juices and wines

Chitinases and thaumatin-like proteins are important grape proteins as they have a great influence on wine quality. The quantification of these proteins in grape juices and wines, along with their purification, is therefore crucial to study their intrinsic characteristics and the exact role they play in wines. The main isoforms of these two proteins from Chardonnay grape juice were thus purified by liquid chromatography. Two fast protein liquid chromatography (FLPC) steps allowed the fractionation and purification of the juice proteins, using cation exchange and hydrophobic interaction media. A further high-performance liquid chromatography (HPLC) step was used to achieve higher purity levels. Fraction assessment was achieved by mass spectrometry. Fraction purity was determined by HPLC to detect the presence of protein contaminants, and by nuclear magnetic resonance (NMR) spectroscopy to detect the presence of organic contaminants. Once pure fractions of lyophilized chitinase and thaumatin-like protein were obtained, ultra-HPLC (UHPLC) and enzyme-linked immunosorbent assay (ELISA) calibration curves were constructed. The quantification of these proteins in different grape juice and wine samples was thus achieved for the first time with both techniques through comparison with the purified protein calibration curve. UHPLC and ELISA showed very consistent results (less than 16% deviation for both proteins) and either could be considered to provide an accurate and reliable quantification of proteins in the oenology field.     

Studies on neurosteroids. Part XIII. Characterization of catechol estrogens in rat brains using liquid chromatography-mass spectrometry-mass spectrometry

The existence of catechol estrogens in rat brains was clarified using liquid chromatography-atmospheric pressure chemical ionization-ion trap-mass spectrometry-mass spectrometry (LC-APCI-MS2). The catechol estrogens were extracted in the presence of ascorbic acid and then derivatized to acetates with acetic anhydride and pyridine. After a successive purification with silica gel mini-column chromatography, reversed-phase solid-phase extraction and preparative HPLC, the obtained fractions containing the catechol estrogen acetates were subjected to LC-APCI-MS2. 2-Hydroxyestrone, 2-hydroxyestradiol and their 4-hydroxy isomers were identified as acetates by comparison with authentic samples based on their chromatographic behavior and mass spectral data. The derivatization to acetate was useful for the treatment of labile catechol estrogens.     

Separation of tryptic phosphopeptides of ribosomal origin by reversed-phase high-performance liquid chromatography

Phosphorylation sites for cyclic AMP-dependent kinase in ribosomal proteins and their synthetic analogues were converted to tryptic phosphopeptides and analysed by reversed-phase high-performance liquid chromatography (RP-HPLC) using gradients of acetonitrile in water and 0.1% trifluoroacetic acid. Tryptic variants differing by only NH2-terminal basic amino acid residues or phosphoryl groups were not always well resolved under these conditions. The different phospho forms could be resolved by RP-HPLC in phosphate buffers at pH 7.0. A combination of gel permeation chromatography, RP-HPLC and thin-layer cellulose mapping was found to be the most effective strategy for the absolute purification of tryptic phosphopeptides from crude tryptic digests.     

Electrophoresis and chromatography of wheat proteins: available methods, and procedures for statistical evaluation of the data.

Analysis of gluten proteins from the wheat grain endosperm has long challenged the analytical chemist. Several hundred unique polypeptides are present, many in large polymers. This complexity, plus useful relationships of composition to genotype and quality, encouraged development and application of electrophoresis and chromatography for gluten analysis. We review the methods of polyacrylamide gel electrophoresis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, isoelectric focusing and high-performance liquid chromatography available for study of wheat proteins. Singly and in combination, they provide rapid, reproducible, high-resolution separations based on size, charge, or surface hydrophobicity. As challenging and important as the analyses themselves, however, is interpretation of data. Subjective evaluation is sometimes possible, but statistical methods such as similarity scores, clustering, principal components, multiple linear regression, and partial least squares now are increasingly used for data analysis. We review the use of these procedures, and precautions necessary to avoid misinterpretation of data. Optimal evaluation of protein analytical data will enhance the value of such analyses in wheat breeding, marketing, and processing.     

Deliberate manipulation of the surface hydrophobicity of an adsorbent for an efficient purification of a giant molecule with multiple subunits

Hydrophobic interaction chromatography (HIC) is often an inevitable step for a satisfying purification in giant vaccine molecules production. But great mass and activity loss associated with poor purity often occur simultaneously. In this paper, high purity and high bioactivity recovery for the HIC process of hepatitis B surface antigen (rHBsAg) purification were achieved through manipulation of surface hydrophobicity of the adsorbent. Spacer arm length and ligand density were regulated, respectively, through which the interaction between the vaccine and the adsorbent was manipulated deliberately. It was found even in a narrow scope, varying spacer arm length and ligand density resulted in purification factor changing from 1 to 96.5, and rHBsAg recovery from 3 to 91%. The optimal purification performance was achieved when the spacer arm was C8 and the ligand density was 9.2 μmol/g suction-dried wet gel with an average distance of ligands of 3.6 nm. This deliberate regulation strategy represents a new approach of improving purification of giant multi-subunit proteins.     

Isolation of Escherichia coli inner membranes by metal affinity two-phase partitioning

As reduction of sample complexity is a central issue in membrane proteomic research, the need for new pre-fractionation methods is significant. Here we present a method for fast and efficient enrichment of Escherichia coli inner membranes expressing a His-tagged integral membrane L-fucose-proton symporter (FucP). An enriched inner membrane fraction was obtained from a crude membrane mixture using affinity two-phase partitioning in combination with nickel-nitrilotriacetic acid (Ni-NTA) immobilized on agarose beads. Due to interaction between the beads and FucP, inner membranes were selectively partitioned to the bottom phase of a polymer/polymer aqueous two-phase system consisting of poly(ethylene glycol) (PEG) and dextran. The partitioning of membranes was monitored by assaying the activity of an inner membrane marker protein and measuring the total protein content in both phases. The enrichment of inner membrane proteins in the dextran phase was also investigated by proteomic methodology, including sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), trypsin digestion and liquid chromatography in combination with tandem mass spectrometry (LC-MS/MS). Using a high level of significance (99.95%) in the subsequent database search, 36 proteins assigned to the inner membrane were identified in the bottom phase, compared to 29 when using the standard sucrose gradient centrifugation method for inner membrane isolation. Furthermore, metal affinity two-phase partitioning was up to 10 times faster than sucrose gradient centrifugation. The separation conditions in these model experiments provide a basis for the selective isolation of E. coli membranes expressing His-tagged proteins and can therefore facilitate research on such membrane proteomes.     

Production and purification of bacterial membrane vesicles for biotechnology applications: Challenges and opportunities

Bacterial membrane vesicles (BMVs) are bi-layered nanostructures derived from Gram-negative and Gram-positive bacteria. Among other pathophysiological roles, BMVs are critical messengers in intercellular communication. As a result, BMVs are emerging as a promising technology for the development of numerous therapeutic applications. Despite the remarkable progress in unveiling BMV biology and functions in recent years, their successful isolation and purification have been limited. Several challenges related to vesicle purity, yield, and scalability severely hamper the further development of BMVs for biotechnology and clinical applications. This review focuses on the current technologies and methodologies used in BMV production and purification, such as ultracentrifugation, density-gradient centrifugation, size-exclusion chromatography, ultrafiltration, and precipitation. We also discuss the current challenges related to BMV isolation, large-scale production, storage, and stability that limit their application. More importantly, the present work explains the most recent strategies proposed for overcoming those challenges. Finally, we summarize the ongoing applications of BMVs in the biotechnological field.     

Analysis and purification of DNA restriction fragments by high-performance liquid chromatography.

The purification and analysis of restriction fragments play a very important role in molecular biology but the traditional assay methods of DNA fragments, based on gel electrophoresis and caesium chloride gradient centrifugation, are time-consuming and difficult to quantify. High-performance liquid chromatography provides an alternative method which allows the direct quantitation of picogram amounts of eluents in short time. In the present work we report the separation of different restriction fragments, the purification of some fragments and the relationship between the length of double-stranded DNA fragments and peak areas.     

High-resolution separation of molybdate-stabilized progestin receptors using high-performance liquid chromatography

Molecular heterogeneity of the human uterine progestin receptor was investigated employing sucrose density gradient centrifugation and high-performance liquid chromatography (HPLC) in size-exclusion (HPSEC), ion-exchange (HPIEC) and chromatofocusing (HPCF) modes. Synthetic progestomimetic ligands, [3H]R5020 and [3H]ORG-2058, were used to identify these receptors. Rapid centrifugation with a vertical tube rotor showed both 8-9 S and 4-5 S receptor species in the presence of 10 mM sodium molybdate with a 90-96% recovery. [3H]R5020 displayed greater nonspecific binding than [3H]ORG-2058. When separated receptor preparations were labeled, each with a different ligand, mixed and separated on optimized gradients, at least two receptor isoforms were identified in the components sedimenting at 8-9 S. HPSEC confirmed the presence of receptor isoforms displaying different molecular size and shape dependent upon the progestin ligand used. When the surface charge properties were examined by HPIEC using AX-1000, two distinct species were observed irrespective of the radioactive ligand. The first peak appeared in the void volume similar to the position of free steroid, indicating the possibility of ligand stripping by the column. The second peak bound steroid specifically and eluted with 100 mM phosphate. If either 8-S or 4-S progestin receptors were first separated by gradient centrifugation then by HPIEC, both receptor isoforms eluted with 60 mM phosphate. Re-chromatography of these on HPIEC also gave the isoform eluting at 60 mM phosphate. HPCF of ligand-bound receptors on AX-500 columns also identified one isoform eluting at pH 5.6-6.1. Using a combination of HPLC techniques and sucrose gradient centrifugation, heterogeneity of the progestin receptor has been demonstrated.     

Electrophoretic studies of antimitochondrial antibodies: identification of mitochondrial antigens specific to primary biliary cirrhosis

Mitochondrial inner membrane proteins extracted from beef heart tissue were examined for reactivity to antimitochondrial antibody (AMA) present in sera of patients with primary biliary cirrhosis (PBC) by an immunoblotting technique. Four proteins, which reacted with AMA, had molecular masses of 70 kDa, 50 kDa, 47 kDa and 40 kDa, as defined by their relative mobility (Rf) in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. All sera of 114 PBC patients were positive with at least one and as many as four of the mitochondrial proteins. The major antigenic proteins of mitochondrial inner membrane to which AMA reacts were the 70 kDa and 47 kDa proteins. All PBC sera containing antibodies to the 50 kDa and/or 40 kDa proteins reacted with 70 kDa as well. The isolation of antigen reacting with AMA of PBC is important to warrant further study of AMA and the cause of the disease. The isolation of responsible antigens had been difficult because the four antigens were insoluble. However, the antigen newly found by us, the 36 kDa fragment, obtained by partial trypsin digestion, is soluble. Using several procedures, the antigenic protein target of AMA was purified from mitochondria for the first time. We determined the N-terminal sequence of the soluble 36 kDa fragment, 25 residues in length. Until now the N-terminal sequence of the 36 kDa protein has not shown significant homology with any known protein. The present results of antigen purification would contribute to the elucidation of the epitopes of AMA antigen.     

Analysis of proteins from a glioma cell line by using micro-scale solution isoelectric focusing in combination with liquid chromatography/tandem mass spectrometry

In this study we have investigated whether micro-solution isoelectric focusing (microsol-IEF) can be used as a pre-fractionation step prior to liquid chromatography/tandem mass spectrometry (LC/MS/MS) and if extensive sample purification of the different fractions is required. We found that, in spite of the high concentrations of buffer and detergents, no clean up of the digested microsol-IEF fractions was necessary before analysis by LC/MS/MS. We also concluded that it is possible to identify at least twice as many proteins in a glioma cell lysate with the combination of microsol-IEF and LC/MS/MS than with LC/MS/MS alone. Furthermore, most of the proteins that were identified from one microsol-IEF fraction by using analytical narrow-range two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and peptide mass fingerprinting with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) were also identified by LC/MS/MS. Finally, we used the combination of microsol-IEF and LC/MS/MS to compare two sample preparation methods for glioma cells and found that several nuclear, mitochondria, and endoplasmic reticulum proteins were only present in the sample that had been subjected to lipid extraction by incubating the homogenized cells in chloroform/methanol/water.     

Phosphopeptide quantitation using amine-reactive isobaric tagging reagents and tandem mass spectrometry: application to proteins isolated by gel electrophoresis

Polyacrylamide gel electrophoresis is widely used for protein separation and it is frequently the final step in protein purification in biochemistry and proteomics. Using a commercially available amine-reactive isobaric tagging reagent (iTRAQ) and mass spectrometry we obtained reproducible, quantitative data from peptides derived by tryptic in-gel digestion of proteins and phosphoproteins. The protocol combines optimized reaction conditions, miniaturized peptide handling techniques and tandem mass spectrometry to quantify low- to sub-picomole amounts of (phospho)proteins that were isolated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Immobilized metal affinity chromatography (FeIII-IMAC) was efficient for removal of excess reagents and for enrichment of derivatized phosphopeptides prior to matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis. Phosphopeptide abundance was determined by liquid chromatography/tandem mass (LC/MS/MS) using either MALDI time-of-flight/time-of-flight (TOF/TOF) MS/MS or electrospray ionization quadrupole time-of-flight (ESI-QTOF) MS/MS instruments. Chemically labeled isobaric phosphopeptides, differing only by the position of the phosphate group, were distinguished and characterized by LC/MS/MS based on their LC elution profile and distinct MS/MS spectra. We expect this quantitative mass spectrometry method to be suitable for systematic, comparative analysis of molecular variants of proteins isolated by gel electrophoresis.     

Monolithic beds of artificial gel antibodies

The introductions of the continuous beds, now often called monoliths [S. Hjertén, J.-L. Liao, R. Zhang, J. Chromatogr. 473 (1989), 273-275] and the artificial, highly selective gel antibodies against antigens as large as proteins, viruses and cells [J.-L. Liao, Y. Wang, S. Hjertén, Chromatographia 42 (1996), 259-262] were breakthroughs in the design of chromatographic beds. This paper deals with a combination of these two methods, i.e., the artificial gel antibodies have been synthesized in the monolithic mode. As antigen we have used human hemoglobin. A comparison of the ion-exchange chromatograms of the eluates from the monolithic columns shows that the monolith prepared in the presence of hemoglobin adsorbed this protein, but not the other proteins in the sample (ribonuclease A and cytochrome c), i.e., this monolith was selective for hemoglobin, whereas the blank column (prepared in the absence of hemoglobin) had no selective properties, since none of the applied proteins were adsorbed. The diameter of the column was 6mm, but the same approach to synthesize a monolithic selective bed can very likely also be used for capillaries and microchips.     

Determination of Pesticides in the Rhizome of Traditional Chinese Medicines by Gas Chromatography with Electron Capture Detection

A simple and efficient gas chromatography method for the determination of 27 organochlorine and pyrethroid pesticides in traditional Chinese medicine was developed. Pesticide residues were extracted from the samples by oscillation and ultrasound, and purified by gel permeation chromatography and florisil solid phase extraction. The analysis was performed by gas chromatography with micro-electron capture detection. The purification conditions, such as the extraction and elution solvents, extraction methodology, and gel permeation chromatography conditions, were optimized. Traditional Chinese medicines were purified by different methods based on their composition. Under optimized conditions, the calibration graph was linear from 10 to 500 µg/L and the correlation coefficients varied from 0.9958 to 0.9997. The detection limits of these pesticides were between 0.09 and 1.82 µg/kg. The recovery of the pesticides spiked in various samples at four concentrations ranged from 73.2% to 106.4%, with relative standard deviations between 1.0% and 14.0%. This study indicates that the method is useful for determining pesticides in rhizome traditional Chinese medicines.     

Profiling of lipids in Leishmania donovani using hydrophilic interaction chromatography in combination with Fourier transform mass spectrometry

There is evidence from our current research on resistance to stibigluconate and from some previous observations that lipid composition may be altered in resistant Leishmania donovani and in order to explore this we required a comprehensive lipidomics method. Phospholipids can be analysed by direct infusion into a mass spectrometer and such methods can work very well. However, chromatographic methods can also be very effective and are extensively used. They potentially avoid ion suppression effects, associate lipid classes with a retention time range and deliver good quantitative accuracy. In the current study three chromatography columns were compared for their ability to separate different classes of lipid. Butylsilane (C‐4), Zic‐HILIC and a silica gel column were compared. The best results were obtained with a silica gel column used in hydrophilic interaction chromatography (HILIC) mode with a mobile phase gradient consisting of (A) 20% isopropyl alcohol (IPA) in acetonitrile (v/v) and (B) 20% IPA in 0.02 M ammonium formate. Using these conditions separate peaks were obtained for triglycerides (TG), phosphoinositols (PI), inositol phosphoceramides (IPC), phosphatidylethanolamines (PE), phosphatidylserines (PS), phosphatidylcholines (PC), sphingosines (SG), lysophosphatidyethanolamines (LPE) and lysophosphatidylcholines (LPC). The methodology was applied to the analysis of lipid extracts from Leishmania donovani and by coupling the chromatography with an LTQ Orbitrap mass spectrometer. It was possible to detect 188 lipid species in the extracts with the following breakdown: PC 59, PE 38, TG 35, PI 20, CPI 13, LPC 11, LPE 2 and SG 10. The fatty acid composition of the more abundant lipids was characterised by MS² and MS³ experiments carried out by using an LCQ Deca low‐resolution ion trap instrument coupled with the silica gel column. The separation of lipids into well‐defined groups gives extra confidence in their identification and minimises the risk of ion suppression effects. High‐resolution mass spectrometry was necessary in order to be able to differentiate between acyl‐ and acyl‐alkyl‐lipids. Copyright © 2010 John Wiley & Sons, Ltd.