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PubMed2006

Novel strategy of high-abundance protein depletion using multidimensional liquid chromatography.

In this study, for the first time, a comprehensive two-dimensional (2D) liquid-phase separation system, coupling strong cation exchange chromatography (SCX) to reversed-phase high performance liquid chromatography (RPLC), instead of specificity depletion method, was developed at the intact protein level for depletion of high-abundance proteins from rat liver. Proteins were prefractionated by SCX in the first dimensional separation, followed by RPLC with high resolution separation. UV absorption intensity was used to differentiate high-abundance proteins. The proteins with the absorbance intensity above 0.1 AU were defined as high abundance proteins and depleted. After removal of high-abundance proteins; other proteins were pooled, digested, and subsequently separated by capillary liquid chromatography coupled with MALDI-TOF/TOF mass spectrometry analysis. The high efficiency of the strategy was demonstrated by analyzing the soluble protein extracted from rat liver tissue. In total, 77 high-abundance proteins were depleted in one experiment flow. The ratio of depleted content of high-abundance proteins to that of total proteins was about 34.5%. In total, 1530 proteins were identified using the depletion strategy. Quantitative estimation of high-abundance proteins through liquid chromatography combined with UV absorption spectra was achieved. On the basis of the reproducible experimental results, a rapid and high-throughput depletion protocol was put forward. Along with depletion of the most (79.1%) high-abundance proteins and the separation of digested peptides, the total separation time could be less than 30 h. This strategy has no bias for depleting high-abundance proteins and enhances the number of identified proteins; therefore, it can be widely used in the global proteins analysis.

Animals↗
PubMed2007

Protein phosphorylation and expression profiling by Yin-yang multidimensional liquid chromatography (Yin-yang MDLC) mass spectrometry.

A system which consisted of multidimensional liquid chromatography (Yin-yang MDLC) coupled with mass spectrometry was used for the identification of peptides and phosphopeptides. The multidimensional liquid chromatography combines the strong-cation exchange (SCX), strong-anion exchange (SAX), and reverse-phase methods for the separation. Protein digests were first loaded on an SCX column. The flow-through peptides from SCX were collected and further loaded on an SAX column. Both columns were eluted by offline pH steps, and the collected fractions were identified by reverse-phase liquid chromatography tandem mass spectrometry. Comprehensive peptide identification was achieved by the Yin-yang MDLC-MS/MS for a 1 mg mouse liver. In total, 14 105 unique peptides were identified with high confidence, including 13 256 unmodified peptides and 849 phosphopeptides with 809 phosphorylated sites. The SCX and SAX in the Yin-Yang system displayed complementary features of binding and separation for peptides. When coupled with reverse-phase liquid chromatography mass spectrometry, the SAX-based method can detect more extremely acidic (pI < 4.0) and phosphorylated peptides, while the SCX-based method detects more relatively basic peptides (pI > 4.0). In total, 134 groups of phosphorylated peptide isoforms were obtained, with common peptide sequences but different phosphorylated states. This unbiased profiling of protein expression and phosphorylation provides a powerful approach to probe protein dynamics, without using any prefractionation and chemical derivation.

Amino Acid Sequence↗
PubMed1980

Resolution of protein disulphide-isomerase and glutathione-insulin transhydrogenase activities by covalent chromatography.

1. Protein disulphide-isomerase (EC 5.3.4.1) and glutathione-insulin transhydrogenase (EC 1.8.4.2) were resolved by covalent chromatography. Both activities, in a partially purified preparation from bovine liver, bind covalently as mixed disulphides to activated thiopropyl-Sepharose 6B, in a new stepwise elution procedure protein disulphide-isomerase is displaced in mildly reducing conditions whereas glutathione-insulin transhydrogenase is only displaced by more extreme reducing conditions. 2. This together with evidence for partial resolution of the two activities by ion-exchange chromatography, conclusively establishes that the two activities are not alternative activities of a single bovine liver enzyme. 3. Protein disulphide-isomerase, partially purified by a published procedure, has now been further purified by covalent chromatography and ion-exchange chromatography. The final material is 560-fold purified relative to a bovine liver homogenate; it has barely detectable glutathione-insulin transhydrogenase activity. 4. The purified protein disulphide-isomerase shows a single major band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis corresponding to a mol.wt. of 57000. 5. The purified protein disulphide-isomerase has Km values for 'scrambled' ribonuclease and dithiothreitol of 23 microgram/ml and 5.4 microM respectively and has a sharp pH optimum at 7.5. The enzyme has a broad thiol-specificity, and several monothiols, at 1mM, can replace dithiothreitol. 6. The purified protein disulphide-isomerase is completely inactivated after incubation with a 2-3 fold molar excess of iodoacetate. The enzyme is also significantly inhibited by low concentrations of Cd2+ ions. These findings strongly suggest the existence of a vicinal dithiol group essential for enzyme activity. 7. When a range of thiols were used as co-substrates for protein disulphide-isomerase activity, the activities were found to co-purify quantitatively, implying the presence of a single protein disulphide-isomerase of broad thiol-specificity. Glutathione-disulphide transhydrogenase activities, assayed with a range of disulphide compounds, did not co-purify quantitatively.

Animals↗
PubMed1989

Purification of the food-borne carcinogens 2-amino-3-methylimidazo [4,5-f]quinoline and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline in heated meat products by immunoaffinity chromatography.

A rapid and simple scheme has been developed for the isolation and purification of two of the major mutagenic heterocyclic amines formed in heated beef products by affinity chromatography using monoclonal antibodies which recognize 2-amino-3-methylimidazo[4,5-f]quinoline (IQ). Two cell lines producing IgG antibodies were established following fusion of Sp2 or P3x.63 myeloma cells with spleen cells of immunized BALB/cby mice. The antigen was bovine gamma globulin haptenized with 2-(3-carboxypropylthio)-3-methylimidazo-[4,5-f]quinoline. The antibodies were immobilized on CNBr-activated Sepharose 4B. IQ and MeIQx formed in heated beef products were partially purified by XAD-2 chromatography and then applied to the affinity columns. Purification by affinity chromatography was adequate for subsequent quantitative analysis by HPLC with UV detection. With this purification scheme as little as 1 g of beef extract or 15 g of fried beef could be assayed for IQ and MeIQx at the part per billion level. Both antibodies had similar affinity constants for IQ (9.3 X 10(6) and 6.7 X 10(6) M-1) and for MeIQx (7.1 X 10(5) and 2.7 X 10(5) M-1) and both were suitable for immunoaffinity purification of IQ from complex mixtures. MAb2 could be used as well to selectively remove MeIQx from meat products after partial purification by XAD-2. MAb1, despite having a 3-fold higher affinity than MAb2 for MeIQx, could not be used for affinity chromatography for this mutagen.

Animals↗
PubMed1989

Purification and characterisation of Clostridium difficile toxin A by bovine thyroglobulin affinity chromatography and dissociation in denaturing conditions with or without reduction.

Highly purified toxin A of Clostridium difficile was obtained by bovine thyroglobulin affinity chromatography followed by two sequential anion-exchange chromatography steps on Q Sepharose FF and Mono Q. After Q Sepharose FF chromatography of a thyroglobulin affinity-purified toxin A preparation, two major peaks of cytotoxicity representing toxins A and B were detected. The homogeneity of the final toxin A preparation obtained from Mono Q anion-exchange fast protein liquid chromatography was ascertained by gel electrophoresis developed by silver stain. The mol. wt of toxin A in non-denaturing conditions was estimated to be 520-540 Kda by native polyacrylamide gel electrophoresis (PAGE) developed by silver stain. In contrast, with sodium dodecyl sulphate (SDS)-PAGE under reducing or non-reducing conditions, a major band of 240 Kda and 10 minor and 27 faint bands (non-reduced conditions), or four minor and 31 faint bands (reduced conditions) were detected after silver staining. In two-dimensional PAGE, the seven minor bands of greater than 240 Kda obtained by non-reducing SDS-PAGE migrated to the 240-Kda position after reduction with beta-mercaptoethanol.

Animals↗
PubMed1982

Analysis of mycolic acids from a group of corynebacteria by capillary gas chromatography and mass spectrometry.

The cell wall of leprosy-derived corynebacteria (a group of 'diphtheroids' isolated from human leprosy lesions and patients' blood) was previously shown to contain, in addition to peptidoglycan and arabinogalactan, mycolic acids. These alpha-branched beta-hydroxy fatty acids were attributed to the corynomycolic group, according to their RF in monodimensional thin-layer chromatography. In the present work, mycolic acids from leprosy-derived and reference corynebacteria have been fractionated by monodimensional and bidimensional thin-layer chromatography and by gas chromatography. Pyrolyzed mycolic acids have been analyzed on conventional packed columns, whereas intact methyl esters of mycolic acids with free and silylated beta-hydroxyl group have been analyzed on capillary columns, and their structure has been established by mass spectrometry. In all leprosy-derived corynebacteria, some 20 components containing 24-36 carbon atoms and 0-4 double bonds were obtained. The three major groups had 32, 34 and 36 carbons, and the frequency of unsaturated versus saturated chains increased proportionally to the molecular weight. For comparison, the main components of a reference corynebacterium. Corynebacterium diphtheriae PW8, had 30 and 32 carbons, and their hydrocarbon chains were essentially saturated. This work confirms the relative chemical homogeneity of different leprosy-derived corynebacteria and describes some peculiar traits in the chemical structure of this group of organisms. In addition, it shows the complexity of the mycolic acid fraction of corynebacterial cell wall and suggests that the mycolic acid pattern is a sort of fingerprint of each bacterial strain grown under standard conditions. Finally, the fractionation of intact corynomycolic acid methyl esters with free or silylated beta-hydroxyl group by capillary gas chromatography proved to be the best analytical procedure at present available for resolving this complex mixture of corynomycolate isomers. Structural determination of silylated samples by mass spectrometry is preferred because they have more diagnostic fragments.

Cell Wall↗
PubMed1990

Selective purification of sex-influenced esterase from rat serum by immunoaffinity chromatographies.

Sex-influenced esterase (ES-SI) is a female-specific serum esterase of rats. There are two alleles at the Es-Si locus, Es-Sia coding for ES-SI and Es-Sib, a silent allele. ES-SI was purified to electrophoretic homogeneity from the serum of adult female SI3 rats by a four-step purification. First, albumin was removed from the serum by Blue-Sepharose CL-6B affinity chromatography. Sera of rats carrying Es-Sia and Es-Sib were utilized to make two novel immunoaffinity columns. Rat anti-(ES-SI) alloantibody was obtained after the immunization of rats carrying Es-Sib with rat ES-SI-positive serum. ES-SI bound to the gel in this antibody-conjugated affinity chromatography. Simultaneously, rabbit anti-(rat serum protein lacking ES-SI) antibody was raised against ES-SI-negative rat serum and used to exclude other serum proteins. ES-SI was recovered in the flow-through fractions in this antibody-conjugated immunoaffinity chromatography, whereas the other serum proteins bound to the gel. ES-SI was selectively purified about 240-fold by combining these immunoaffinity chromatographies. This combination is a very powerful technique for purification of the protein. Finally, ES-SI was purified by preparative polyacrylamide gel electrophoresis after which it appeared as a single protein band (using silver staining) on SDS/polyacrylamide gel electrophoresis with a molecular mass of 72 kDa. In the ordinary estimation of enzyme purification, the purification of ES-SI was 30-fold at most, because the specific activity of the enzyme was calculated from the bulky esterase activity. However, ES-SI was selectively purified 2280-fold comparing its recovery to that of total protein. ES-SI exhibited a pI of 4.6 and optimum pH of 6.7. The activity of ES-SI was completely inhibited by diisopropyl fluorophosphate at 10 nM but not at all by eserine.

Animals↗
PubMed1990

Purification of Staphylococcus aureus beta-lactamases by using sequential cation-exchange and affinity chromatography.

Boronic acids are active-site inhibitors of serine beta-lactamases, and a phenylboronic acid-agarose affinity column has been used to purify beta-lactamase from crude cell extracts of several bacterial species. We applied phenylboronic acid-agarose chromatography to the purification of Staphylococcus aureus beta-lactamase. Two factors interfered with the success of the previously described single-step chromatographic protocol. First, staphylococcal beta-lactamase exhibited non-active-site-mediated adsorption to the agarose used as a support for the meta-aminophenylborate ligand, preventing the recovery of beta-lactamase from the column. Second, the staphylococcal beta-lactamases exhibited low affinity for meta-aminophenylborate with inhibition constants (Kis) ranging from 8.0 x 10(-3) to 20.0 x 10(-3) M. These problems were resolved by modifying the buffers utilized during chromatography and increasing the dimensions of the affinity column, and a two-stage procedure consisting of cation-exchange chromatography followed by affinity chromatography was used to purify each of the four variants of staphylococcal beta-lactamase. The mean specific activities of the purified type A, B, C, and D beta-lactamases were 44.6, 12.2, 10.6, and 30.8 mumol of nitrocefin hydrolyzed per min/mg of protein, respectively. Dimer formation, presumably from intramolecular cysteine-cysteine cross-linking, was observed with the type D beta-lactamase but not with the type A, B, or C enzyme.

Boronic Acids↗
PubMed1974

Paper chromatography as an adjunct in the identification of anaerobic bacteria.

Modified paper chromatography procedures for the analysis of fatty acids produced by anaerobic bacteria are described. Both ethylamine and hydroxylamine derivatives of fatty acids were prepared from inoculated anaerobic culture broth. The derivatives were spotted on chromatography paper and developed with appropriate solvents. Paper chromatography is a valuable alternative to gas liquid chromatography as an ancillary procedure in the identification of anaerobic bacteria in the clinical bacteriology laboratory.

Acetates↗
PubMed1982

Analysis of short-chain acids from anaerobic bacteria by high-performance liquid chromatography.

A standard mixture of 25 short-chain fatty acids was resolved by high-performance liquid chromatography, using an Aminex HPX-87 column. The acids produced in culture media by anaerobic bacteria were analyzed by high-performance liquid chromatography after extraction with ether and reextraction into a small volume of 0.1 N NaOH. The presence of fumaric acid in culture extracts of Peptostreptococcus anaerobius was confirmed by gas chromatography-mass spectrometry analysis of the trapped eluent fractions from the high-performance liquid chromatography column.

Bacteroides fragilis↗
PubMedDate not supplied

Globin chain synthesis analysis by high performance liquid chromatography in the screening of thalassemia syndromes.

We applied reversed phase high performance liquid chromatography for globin chain synthesis analysis in screening for beta-thalassemia. The alpha/non-alpha-globin chain synthesis ratios have been determined in alpha-, beta-, and deltabeta-thalassemia carriers using the classical carboxymethyl cellulose chromatography as the reference method. Reversed phase high performance liquid chromatography is fast, accurate, and reproducible, and may be a suitable alternative for the traditional carboxymethyl cellulose chromatography.

Carboxymethylcellulose Sodium↗
PubMed2005

[The method development of subcritical water chromatography].

Subcritical water is formed from pure water under high temperature and high pressure. The change of hydrogen-bond network results in the obvious change of physical properties of water such as polarity and viscosity. Subcritical water chromatography (SubWC), as a method of high performance liquid chromatography which takes subcritical water as mobile phase, is one kind of novel separation technique developed recently. Common gas chromatographic or liquid chromatographic instrument modified properly can be used as the instrument for SubWC. Both packed columns used in liquid chromatography and capillary columns used in gas chromatography can be applied in SubWC. The selectivity can be adjusted by changing column temperature or pressure and addition of the organic modifiers or salts in mobile phase. Both flame ionization detector and ultraviolet detector can be used as the detecting part of SubWC. Thus the range of its application and selection of optimal conditions are largely broadened. Currently this novel separation mode is still in the stage of research and development and mostly used in the fast separation of samples with polar and medium polar. The characteristics of SubWC are illustrated from the aspects and design of instruments, the elution characteristics of mobile phase and the selection of stationary phase. Moreover, the recent development of SubWC is systematically reviewed.

Chromatography, High Pressure Liquid↗
PubMed2006

[Isolation and purification of the process impurity in losartan by reversed-phase column chromatography].

A process impurity (approximately 4%, w/w) was isolated from losartan crude by low-pressure reversed-phase column chromatography. At first the elution portions containing the target compound were collected. After evaporation and recrystallization, the purity of the target compound was found to be over 99% based on high performance liquid chromatography with ultraviolet absorption. The electrospray ionization-mass spectrometry result showed that it was an isomeric compound of losartan, which had same fragmentation pattern with its parent compound. And at last the compound was further characterized by nuclear magnetic resonance. This research demonstrates that the low-pressure reversed-phase column chromatography can raise separation efficiency and recovery comparing with other conventional column chromatography, and the technique can be applied as an effective means for the enrichment and extraction of impurities in pharmaceutical separation.

Adsorption↗
PubMed1990

[The use of liquid exclusion chromatography for evaluating methods of treating urologic diseases].

Gel chromatography was used for measuring medium-molecular levels in the blood sera of urologic patients: (1) low-pressure chromatography in Sephadex G-15 packed columns with fractionation range less than 1500 dalton or in Toyopearl HW-40 packed columns with fractionation range from 100 to 100,000 dalton; (2) high-pressure chromatography in LKB (Sweden) blue columns. Liquid exclusion chromatography was employed to assess the efficacy of blood extracorporeal detoxication of urologic patients by means of hemoperfusion, hemodiafiltration, hemodialysis, plasma perfusion, plasmapheresis. The highest correlation between patients' state of health and blood levels of medium molecules was observed in hemodiafiltration.

Chromatography, Gel↗
PubMed1989

Multiresidue procedures for the determination of chlorinated dibenzodioxins and dibenzofurans in a variety of foods using capillary gas chromatography-electron-capture detection.

Multiresidue digestion-extraction procedures for the determination of chlorinated dioxins and furans in a wide variety of products are presented. Procedure selection is dependent upon the residue(s) of interest, and on the fat content of the product. Additional cleanup is accomplished using column chromatography and a Florisil trap. The separation of residues is achieved by fraction collection off of two high-performance liquid chromatographic systems. Capillary gas chromatography employing electron-capture detection is used for quantitation. The extracts are suitable for gas chromatography mass spectrometry or gas chromatography with Hall electrolytic conductivity detection. Results of analysis, recovery data, and interlaboratory comparisons are presented. Spike recoveries will typically average 90% +/- 10%.

Benzofurans↗
PubMed1985

UV-absorbance profile of human leukocytic ultrafiltrate after affinity chromatography on immobilized m-aminophenyl boronic acid: implication for transfer factor purification.

The UV-absorbance of the lysed human leukocyte ultrafiltrate (LLU) was investigated by Sephadex G-15 gel permeation chromatography before and after affinity chromatography on immobilized m-aminophenyl boronic acid. This biospecific fractionation caused substantial changes in 260/280-nm absorbance ratio of individual peaks as revealed by Sephadex G-15 gel permeation chromatography. While the material which had passed through the affinity support unretarded (W) appeared to be partly or completely deprived of peak IV, peaks IV and V were fairly enriched in the retarded material (R). This material, on the other hand, nearly completely missed peaks III, VI and VII. Peak III was copiously represented in material W. Boronate affinity chromatography caused enrichment in the protein content as well as in the content of purine/pyrimidine-bound ribose of material R, i.e. cis-diol group containing (RNA-like) material, considered to represent functional transfer factor activity. The described findings further support the use of immobilized derivatives of boronic acid in the separation of coplanar cis-diol group containing compounds from other constituents of LLU.

Boronic Acids↗
PubMed1985

Purification of angiotensin-converting enzyme from rabbit lung and human plasma by affinity chromatography.

Lisinopril (N alpha-[(S)-1-carboxy-3-phenylpropyl]L-lysyl-L-proline), a potent angiotensin-converting enzyme inhibitor, is an exceptionally selective affinity chromatography ligand for this enzyme. Affinity chromatography furnishes electrophoretically homogeneous enzyme directly from crude homogenates of rabbit lung tissue, a 1,000-fold purification; also, it affords a 100,000-fold enrichment of the more rare human plasma enzyme in a single step. The affinity of angiotensin-converting enzyme for the Sepharose-spacer-lisinopril matrix (Ki matrix = 1 X 10(-5) M) is weak compared to its affinity for free lisinopril (Ki = 1 X 10(-10) M). The capacity of the affinity column is described quantitatively as a function of Ki matrix, lisinopril, and enzyme concentrations. The recovery of bound enzyme is low in chromatography of crude tissue samples (10-40%), although it approaches a reversible process (70-100%) with pure enzyme. The holoenzyme is converted to Zn2+-free apoenzyme to effect removal of lisinopril. In this process, the rate constant for spontaneous dissociation of Zn2+ from free enzyme is 1 X 10(-2) s-1 (t 1/2 = 1 min), which places a lower limit of 3 X 10(-10) M on the dissociation constant of Zn2+ at neutral pH from angiotensin-converting enzyme. The exceptional selectivity of lisinopril as an affinity chromatography ligand for angiotensin-converting enzyme suggests it is among the most specific inhibitors designed for any enzyme.

Animals↗
PubMed1981

Specific thyroxine nuclear receptors: DEAE-sephadex chromatography of rat liver nuclear extract.

Specific binding of thyroxine to protein fraction obtained by DEAE-Sephadex chromatography of dialyzed (0.4 mol 1-1 KCl) nuclear extract of purified rat liver nuclei was studied. A procedure for several-fold purification of thyroxine nuclear receptor included isolation of purified rat liver nuclei (by centrifugation through 1.7 mol 1-1 sucrose), extraction, dialysis, Sephadex G-100 and DEAE-Sephadex A-50 chromatography. It was found that a protein peak eluting at 0.05 mol 1-1 NaCl does not show any fraction specifically binding thyroxine. However, specific binding of nuclear receptor for thyroxine was found in a peak eluting at 0.20 mol 1-1 NaCl, the capacity being about 600 fmol mg-1 protein. Moreover, total binding capacity of intact rat liver homogenate, nuclei, non-dialyzed and dialyzed (0.4 mol 1-1 KCl) nuclear extract, Sephadex G-100 and DEAE-Sephadex A-50 chromatography protein fractions for thyroxine was tested. A marked increase (about 20 fold) in the total thyroxine binding expressed in fmol mg-1 of protein in DEAE-Sephadex A-50 eluate was found as compared to whole nuclei, nuclear extract or Sephadex G-100 chromatography fractions. Partial fractionation of rat liver nuclear extract combined with thyroxine binding studies demonstrated that rat liver nuclei contain a non-histone protein fraction--a receptor that is responsible for a specific binding of thyroxine.

Animals↗