Thin-layer chromatography-densitometry of minor acidic phospholipids: application to lipids from erythrocytes, liver, and kidney

A method for quantitative determination of acidic phospholipids by thin-layer chromatography (TLC) followed by densitometry is described. The total lipids were separated into neutral and acidic fractions by DEAE-Sephadex column chromatography. A clear-cut separation of acidic phospholipids was achieved by high-performance TLC with a solvent system of chloroform/acetone/acetic acid/formic acid/water (60/60/4/10/3). Each phospholipid band was quantitated by densitometry with the use of an internal standard. The lipid compositions of sheep and mouse erythrocytes and of rat liver and kidney were determined by the present method.     

Separation of naturally occurring 3',5'-cyclic ribonucleotides using high pressure liquid chromatography.

A simple and fairly rapid procedure is described for separation of a mixture of five 3′, 5′-cyclic ribonucleotides. The separation was achieved by high pressure liquid chromatography on a Vydac pellicular anion exchange column using a linear gradient of 0.5 to 1.0 m acetic acid as the eluting solvent.     

Selective separation of tryptophan-containing peptides via hydrophobic modification with 2-nitro-4-carboxyphenylsulfenyl chloride

Selective separation of tryptophan-containing peptides has been attempted using 2-nitro-4-carboxyphenylsulfenyl chloride (NCps)-C1 as a reagent for hydrophobic modification of tryptophan. S-Carboxymethylated proteins were modified with NCps-C1 in 70% acetic acid and digested with TPCK-trypsin, and the digests were fractionated, directly or after partial fractionation on a Sephadex G-25 column, by high performance liquid chromatography using a reverse phase column. The tryptophan-containing peptides from the tryptic digests of S-carboxymethylated hen-egg white lysozyme and Trimeresurus flavoviridis phospholipase A2 were thus selectively separated and the amino acid sequences were determined, showing the validity of the method. The phenylthiohydantoin derivative of 2-(2'-nitro-4'-carboxyphenylthio)-L-tryptophan was synthesized and its spectroscopic and chromatographic properties determined, enabling us to identify the derivative on Edman sequencing.     

Peptide fractionation by high-performance liquid chromatography on an Asahipak GS-320 column: application to determination of the disulfide pairings in ribonuclease F1

High-performance liquid chromatography on an Asahipak GS-320 column using isocratic elution with 0.1 M acetic acid has proven effective for fractionation of peptides of molecular weights lower than 3000. This technique enabled the separation of the peptides derived from digestion of native ribonuclease F1 by trypsin and chymotrypsin in combination with conventional gel filtration through Sephadex G-25 and reversed-phase HPLC. Amino acid analysis of the cystine-containing peptides thus obtained revealed the disulfide linkages Cys-6-Cys-102 and Cys-24-Cys-84 in this protein. The behavior of a number of peptides in the HPLC on an Asahipak GS-320 column is described and the separation mechanism is discussed.     

一种简便的肌醇磷脂微量分析方法

分析磷脂酰肌醇循环(PI cycle)的磷脂组分常采用双向薄层层析法．建立了一个简单快速的单向薄层层析分离肌醇磷脂方法．首先采用不同的有机溶剂体系分别提取非多磷酸肌醇磷脂和多磷酸肌醇磷脂,然后用不同的层析展开体系,对两部分磷脂进行单向薄层层析分离．采用无载体 ³²P标记实验对该方法分离效果进行了观察．此法适用于同位素标记和非标记样品中肌醇磷脂组分的比较分析及多磷酸肌醇磷脂的提取、纯化和定量．     

Optimization of Immobilized Gallium (III) Ion Affinity Chromatography for Selective Binding and Recovery of Phosphopeptides from Protein Digests

Although widely used in proteomics research for the selective enrichment of phosphopeptides from protein digests, immobilized metal-ion affinity chromatography (IMAC) often suffers from low specificity and differential recovery of peptides carrying different numbers of phosphate groups. By systematically evaluating and optimizing different loading, washing, and elution conditions, we have developed an efficient and highly selective procedure for the enrichment of phosphopeptides using a commercially available gallium(III)-IMAC column (PhosphoProfile, Sigma). Phosphopeptide enrichment using the reagents supplied with the column is incomplete and biased toward the recovery and/or detection of smaller, singly phosphorylated peptides. In contrast, elution with base (0.4 M ammonium hydroxide) gives efficient and balanced recovery of both singly and multiply phosphorylated peptides, while loading peptides in a strong acidic solution (1% trifluoracetic acid) further increases selectivity toward phosphopeptides, with minimal carryover of nonphosphorylated peptides. 2,5-Dihydroxybenzoic acid, a matrix commonly used when analyzing phosphopeptides by matrix-assisted laser desorption/ionization mass spectrometry was also evaluated as an additive in loading and eluting solvents. Elution with 50% acetonitrile containing 20 mg/mL dihydroxybenzoic acid and 1% phosphoric acid gave results similar to those obtained using ammonium hydroxide as the eluent, although the latter showed the highest specificity for phosphorylated peptides.     

金属螯合亲和层析纯化金属硫蛋白

将二价铜离子螯合在Chelating Sepharose Fast Flow凝胶上制成亲和层析柱,锌诱导兔肝和镉诱导小鼠肝经匀浆、乙醇处理后上柱,用pH4.0的醋酸盐缓冲液平衡,再用pH5.2不同浓度的醋酸盐缓冲液分别洗脱,可得两个金属硫蛋白(MT)洗脱峰,经确定先后为MT-2和MT-1.分离方法比传统的凝胶过滤-离子交换法简单、省时,适于实验室规模分离纯化.     

Use of Gas-Liquid Chromatography to Determine the End Products of Growth of Lactic Acid Bacteria

A simple gas-liquid chromatographic procedure for analyzing ethanol, acetic acid, acetoin, and racemic and meso-2,3-butylene glycol in broth media is described. Overnight broth cultures were filtered or centrifuged, and the filtrate or supernatant was treated with formic acid to aid separation of volatile fatty acids. Samples were then directly analyzed by gas-liquid chromatography on a 20% Tween 80-Chromosorb W-AW column and propionic acid as an internal standard. A complete analysis took ca. 8 min. The method can be used to distinguish homofermentative from heterofermentative lactic acid bacteria based on the level of ethanol produced and citrate-utilizing from non-citrate-utilizing lactic acid bacteria based on the levels of acetic acid produced. The method also has potential in distinguishing other bacterial fermentations. Of the 13 species of lactic acid bacteria tested, Streptococcus lactis subsp. diacetylactis was the major producer of 2,3-butylene glycol (total range, 0.3 to 3.5 mM), and, except for strain DRC1, both the racemic and meso isomers were produced in approximately equal amounts.     

Chromatography of peptides related to beta-endorphin

Chromatographic procedures are described here for the resolution of beta-endorphin and its related peptides at picomolar concentration. Initially gel filtration is carried out on Sephadex G75 in 50% acetic acid, providing peptides with the approximate molecular size of beta-endorphin. The group of beta-endorphin-related peptides is resolved by ion-exchange chromatography on the pyridinium form of sulfopropyl Sephadex C25 in the presence of 50% acetic acid. The addition of 125I-labeled marker peptides prior to chromatography allows the recovery of each peptide to be calculated and provides a guide for identifying the elution positions of the endogenous peptides. Additional resolution can be obtained by high-pressure liquid chromatography (HPLC) of the sulfoxide forms of the peptides on muBondapak C18 under acidic conditions. The advantages and disadvantages of ion-exchange chromatography and high-pressure liquid chromatography are discussed for the purification of small amounts of basic, hydrophobic peptides.     

Thin-layer separation and quantification of bile acids

A method has been developed for quantification of total free and conjugated bile acids separated on silica gel HR coated thin-layer chromatography plates. Aliquots of bile acid solutions are applied to channeled plates which are developed with either ethyl acetate: isooctane: glacial acetic acid 10:10:2 v/v for free bile acid separation, or chloroform:methanol:glacial acetic acid:water 130:50:4:8 v/v for conjugated bile acid separation. Bile acids are determined directly in serial areas of silica gel by treating gel areas suspended in tris buffer with resazurin reagent. The method is quantitative and as little as 0.1 μg of bile acid is readily determined. Application of the method to determinations of bile acids in crude fecal extracts is described.     

Visualization method for sulfolipids and its application to the determination of nanomole quantities of lipid sulfur

A simple and sensitive visualization method for sulfolipids on a thin-layer chromatogram is described. By spraying with an acidic solution of azure A, a complex was formed between an anionic sulfolipid and a blue cationic compound. After the unbound dye was washed out by brief soaking in methanol, sulfolipids were visualized as clear dark-blue bands on a light-blue background. As little as 0.5 nmol could be detected. Sulfolipid-dye complex was estimated by densitometry or colorimetric measurement after extraction with chloroform/methanol. For the quantitative determination of sulfolipids having long sugar chains, it is necessary to treat thin-layer chromatography plates with acetic anhydride before color development. Of the other tissue lipids not containing sulfuric acid ester that were tested none were stained significantly. A linearity of quantitative determination was observed over the range of 1-8 nmol.     

Pentacyclic triterpenoids and sterols from seven species of mangrove

The isolation of pentacyclic triterpenoids from seven species of fresh mangrove leaves using a simple and rapid method is described. The leaves were homogenized using chloroform—methanol and the extract was diluted with water to precipitate out triterpenoids which were separated into neutral and acidic fractions. These were analysed by gas-liquid chromatography as acetyl and trimethylsilyl ether derivatives on a 3% OV-17 column. Sterols were isolated from the chloroform layer by preparative thin layer chromatography and were analysed by gas-liquid chromatography as their trimethylsilyl ether derivatives on a 3% OV-17 column. The triterpenoids found were α-amyrin, β-amyrin, lupeol, oleanolic acid and ursolic acid in most of the samples. Sterols found in all the samples were cholesterol, campesterol, stigmasterol, sitosterol and stigmast-7-en-3β-ol. Retention indices of the triterpenoids and sterols have been determined.     

Formation of aminosuccinyl peptides during acidolytic deprotection followed by their transformation to piperazine-2,5-dione derivatives in neutral media.

Prolonged acidic treatment of Boc-Leu-Asp(OBut)-Phe-NH2 with 4N HCl in acetic acid resulted in H-Leu-Asc-Phe-NH2 . HCl(Asc, aminosuccinyl), which transformed partially to cyclo[Leu-Asp(Phe-NH2)] during its purification by column chromatography on silica gel with a mixture of ethyl acetate/pyridine/acetic acid/water = 60:20:6:11, i.e. in neutral medium. Examination of the imide formation was extended to different reaction conditions (no imide derivative was detected in trifluoroacetic acid), to several protected derivatives of L-aspartyl-L-phenylalaninamide and to tripeptides containing an aspartyl residue in the middle position. It was clearly demonstrated that in strongly acidic media the imide derivatives are directly formed from the aspartyl peptides containing a free beta-carboxyl group. The influence of the C-terminal residue was greater than the N-terminal on both the rate of formation of the imide and its further transformation to piperazine-2,5-dione derivative. In aqueous ethanol the X-Asc-Y-NH2 (X, Pro, Leu; Y, Gly, Ala, Val, Phg, Phe) containing N-terminal proline are more readily transformed to piperazine-2,5-dione derivatives, but compared to simple proline dipeptides the rate of this transformation is relatively slow because of the crowdedness of the tricyclic transitional state.     

The complete amino acid sequence of a mouse κ light chain

The complete amino acid sequence of the kappa-chain of the mouse myeloma protein MOPC 21 was established. The protein was reduced and alkylated with iodo[2-(14)C]acetic acid, and 21 tryptic peptides were isolated, mainly by paper electrophoresis and paper chromatography. Three large tryptic peptides (of 35, 36 and 42 residues), which were difficult to isolate in this manner, were obtained pure and in excellent yields by a combination of Sephadex G-50 gel filtration in 1% (w/v) NH(4)HCO(3) and chromatography on a DEAE-cellulose column in ammonium acetate buffer, pH8.1. Peptides overlapping the tryptic peptides were isolated from a chymotryptic digest. The chain is 214 residues long. Microheterogeneity of two peptides was observed and is believed to be due to deamidation. It was not excluded that such deamidation could occur in serum from which the protein was isolated. The sequence is compared with the sequences of two other mouse kappa-chains, and with the human kappa-chain basic sequences.     

High-performance liquid chromatographic determination of protoporphyrin and zinc protoporphyrin in blood

Zinc protoporphyrin and protoporphyrin free acid in blood were determined by high-performance liquid chromatography on a C₁₈ column. Results for 63 human blood samples obtained through a lead poisoning detection program compared favorably with the widely-used ethyl acetate—acetic acid extraction determination. Blood from 16 rats which had been maintained on water heavily spiked with chloroform or bromodichloromethane and blood from a lead-poisoned cow were examined by this procedure.     

A radioassay for nonoxidized methionine in peptides. A method for identifying (after isoelectric focusing) and for estimating biologically active forms of corticotropin and other hormones

A radioassay for nonoxidized methionine in peptides is described; it has advantages over other methods currently used because of its simplicity, sensitivity, accuracy, and applicability to individual peptide components in mixtures and to many samples at a time. Methionyl residues were S-carboxymethylated with iodo[2-14C]acetic acid; iodo[2-3H]acetic acid did not provide a stable radioactive tracer. The labeled peptide was isolated by carboxymethylcellulose chromatography or by isoelectric focusing (IEF) or electrophoresis in polyacrylamide gel, and its radioactivity measured. The assay was applied to corticotropins, alpha-melanotropin, bombesin, glucagon, substance P, parathormone, and calcitonin. Twenty-four to thirty samples were conveniently analyzed at a time with a lower detection limit of less than 1 nmol of methionine per sample. The accuracy of the assay, assessed also by reverse-phase high-performance liquid chromatography, is a consequence of its precision, the specificity of the reaction with iodoacetic acid, and the use of an appropriate standard of the peptide being assayed. Methionine was identified, and could be estimated, in individual peptide components of a mixture by using IEF to separate simultaneously the labeled peptide from iodo[2-14C]acetic acid and from other peptide and protein components. This was facilitated by a convenient method for detecting and quantifying these peptides after IEF. The assay is particularly useful for several peptide hormones whose biological activity depends on their sole methionine residue being in a nonoxidized state. It can be used for monitoring their isolation or synthesis and their stability during processing and storage, as well as for evaluating differences in biological potency between preparations and analogues.     

High-pressure liquid chromatography of plasma free acid porphyrins

The separation and quantitation of plasma free acid porphyrins by high-pressure liquid chromatography and fluorescence is described. Porphyrins were extracted from plasma in a simple manner with a recovery >90%. They were separated by high-pressure liquid chromatography on a silica gel (10 μm) column, using a gradient of acetone:dilute acetic acid. Resolution of seven free acid porphyrin standards including coproporphyrins I and III, but not uroporphyrins I and III, was achieved in 12 min at picomolar concentrations. Plasma of patients with erythropoietic protoporphyria displayed protoporphyrin. Uroporphyrin was the only porphyrin found in plasma of eight patients with porphyria cutanea tarda. Normal plasma contained small amounts of uroporphyrin and/or traces of protoporphyrin.     

Purification of synthetic peptides. Immobilized metal ion affinity chromatography (IMAC).

Immobilized metal ion affinity chromatography (IMAC) is a useful method for purification of synthetic peptides with an N-terminal metal-binding amino acid such as His, Trp, or Cys, especially when such residues are not present in other parts of the molecule. In solid phase peptide synthesis (SPPS), capping with acetic anhydride will, in principle, produce truncated peptides as the only side-products due to incomplete couplings. Consequently, only the desired product will carry the affinity label. Most of the impurities, therefore, can be removed by a single passage through an IMAC column. Some representative examples are presented, where fairly large peptides (30-40 amino acid residues) were efficiently purified by this approach.     

Use of a silicic acid microcolumn to assay acyl-CoA: lysophosphatidylcholine acyltransferase

A simple and rapid method for assaying acyl-CoA:lysophosphatidylcholine acyltransferase is described. This method is based on silicic acid microcolumn chromatography using labelled lysophosphatidylcholine (lysoPC) as substrate. The reaction was stopped by conventional Folch extraction. The chloroform extract (2 ml) was deposited on the silica gel and pushed through with air, and then elution was performed with methanol/water (50:50, v/v). Under these conditions, only the labelled phosphatidylcholine (PC) synthesized was retained on the gel, and this was then removed from the column and counted immediately. This method gave enzyme activities comparable to those obtained with the TLC method, and has proved to be reproducible. The new method, however, is both faster and safer than the classical TLC method.