Rapid separation of Escherichia coli 30S ribosomal proteins by fast protein liquid chromatography (FPLC)

The proteins of the 30S ribosomal subunit from Escherichia coli have been separated by reverse-phase high-performance liquid chromatography on a short alkyl chain (C1/C8)-coated phase. The reverse-phase column was connected to a fast protein liquid chromatography (FPLC) system. The 21 proteins of the 30S ribosomal subunit were resolved into 16 peaks. Eleven proteins were isolated in purified form in a single chromatographic run as shown by polyacrylamide gel electrophoresis and amino acid analysis. Interestingly, the retention times of some proteins differed from the retention times observed on other reversed-phase support materials. The results show the speed and resolution of reverse-phase FPLC for both analytical and semi-preparative separations of 30S ribosomal proteins.     

Rapid chromatofocusing of proteins

Chromatofocusing, a chromatographic technique whereby proteins are selectively eluted from an ion-exchange support according to their pI values, has been adapted to high-performance liquid chromatography. It was found that chromatofocusing can detect heterogeneity in protein preparations not demonstrated by reverse-phase or size exclusion chromatography and that the resolution of chromatofocusing is comparable to ion-exchange chromatography. Although chromatofocusing may not resolve proteins as well as isoelectric focusing, it has advantages over this technique in both speed and capacity. The usefulness of chromatofocusing as an additional technique in the analysis and preparation of proteins is discussed. The rapid separation technique described here is able to resolve protein mixtures in the chromatofocusing mode in approximately 30 min.     

Preparative high-performance liquid partition chromatography of proteins.

Methods for preparative high-performance liquid chromatography (hplc) of proteins are described. Both normal and reverse-phase chromatography were studied and adapted to the fractionation of proteins in quantities of up to 50 mg. Lichrosorb Diol was used as a “normal phase” for chromatography of hydrophobic proteins. Lichrosorb RP-8 was used for reversephase chromatography of proteins.     

Separation of the major species of interstitial collagen by reverse-phase high-performance liquid chromatography

Results presented here demonstrate a further application of reverse-phase high-performance liquid chromatography to the separation of large proteins. At a pH near 4.5 with a high pyridine concentration, we have completely separated three major types of human collagen (Types I, II, and III) from mixtures. We illustrate the application of this technique to the preparation of Types I and II collagen from lathyritic chick cartilage extracts.     

Peptide binding domains determined through chemical modification of the side-chain functional groups.

A clear understanding of the specific secondary structure and binding domain resulting from the interactions of proteins and peptides with lipid surfaces will provide insight into the specific functions of biologically active molecules. We have shown in earlier studies that the stationary phases used in reverse-phase high-performance liquid chromatography represent a model artificial lipid surface for the study of induced conformational states of peptides on lipid interaction. We have now used reverse-phase high-performance liquid chromatography to determine the binding domains of peptides and, by extension, of proteins to a lipid surface. This approach consists of performing chemical modifications of specific amino acid side-chain functionalities after the interaction of the peptides with the reverse-phase high-performance liquid chromatography C18 groups. The susceptibility to oxidation was also studied after binding of the same peptides to liposomes. Oxidation of a single methionine residue "walked" through an amphipathic alpha-helical 18-mer peptide was selected to illustrate this approach. The extent of oxidation was found to be clearly dictated by the accessibility of the methionine residue to the aqueous mobile phase. The binding domain found for the peptide in its lipid-induced conformational state was unequivocally the entire hydrophobic face of the amphipathic alpha-helix.     

Separation of o-Phthaldialdehyde Derivatives of Free Amino Acids from Plant Tissues by Isocratic Reverse-phase High-performance Liquid Chromatography

A reverse-phase high-performance liquid chromatography procedurehas been developed for rapid separation and quantitation offree amino acids as o-phthaldialdehyde derivatives. A two stepisocratic solvent system was used which enabled an accurateanalysis at nanomole level. However, two major disadvantagesto this procedure were the lack of reaction of proline and theco-elutions of threonine/glycine and tryptophan/methionine.The free amino acids in Zea mays roots were separated by usingthe method described. Amino acids, liquid chromatography, o-phthaldialdehyde derivatives, reverse-phase chromatography, Zea mays L, maize, corn     

A rapid purification procedure for tetrodotoxin derivatives by high-performance liquid chromatography

A simple procedure for purification of tetrodotoxin (TTX) derivatives by high-performance liquid chromatography is described. Chemically oxidized TTX, C₁₁-nortetrodotoxin (nor-TTX), was purified and collected by reverse-phase chromatography. The separation of nor-TTX from unreacted TTX was excellent and recovery of nor-TTX was more than 90%. The isolated nor-TTX was further coupled with lysine, and the coupled product was purified again by high-performance liquid chromatography on a cation-exchange column. The separation of all compounds required less than 15 min. The uv monitoring at 230 nm allowed the detection of TTX derivatives at the 2- to 3-ng level.     

High-performance liquid chromatographic analysis of demethylmenaquinone and menaquinone mixtures from bacteria

Demethylmenaquinone and menaquinone mixtures from some species of enterobacteria were analysed by reverse-phase partition high-performance liquid chromatography. This method allowed clear separation and quantitative determination of these quinone components.     

A simple and rapid method of quantitative analysis of phosphoamino acids by high-performance liquid chromatography

A high-performance liquid chromatographic system for the separation of nonradiolabeled phosphoamino acids and orthophosphate by ion-pair reverse-phase chromatography has been developed. By the use of low-ionic-strength phthalate buffers at pH 6.3, the phosphoamino acids can be visualized by virtue of this uv-active eluant. The technique is sensitive to 200 pmol of phosphoamino acid and has been shown to be directly applicable to the analysis of isolated phosphoproteins.     

High-performance liquid chromatographic analysis of demethylmenaquinone and menaquinone mixtures from bacteria

Demethylmenaquinone and menaquinone mixtures from some species of enterobacteria were analysed by reverse-phase partition high-performance liquid chromatography. This method allowed clear separation and quantitative determination of these quinone components.     

Postcolumn detection of serum proteins with the biuret and Lowry reactions

The Lowry and biuret reactions have been adapted for the selective detection of chromatographically resolved proteins, specifically proteins separated by high-performance liquid chromatography. The protein reagents are continuously added to the column effluent and produce the characteristic chromophores with both proteins and peptides. The reaction chemistries are compatible with ion-exchange, steric exclusion, and reverse-phase chromatography. Detection limits for proteins resolved by ion-exchange are about 5 to 10 micrograms with the Lowry reaction. Peptides containing tyrosine can be detected at the 100-ng level when chromatographed on reverse-phase columns. The biuret reaction is about 8 times less sensitive for proteins and not very effective for peptides. Reaction detection can be combined with direct absorbance detection in the uv to distinguish proteinaceous peaks from other peaks containing uv-absorbing compounds.     

Separation of peptides by reverse-phase high-performance liquid chromatography using propyl- and cyanopropylsilyl supports

The separation of peptides and proteins by reverse-phase high-performance liquid chromatography with cyanopropylsilyl and large-pore propylsilyl supports, together with aqueous trifluoroacetic acid/acetonitrile gradients, was studied. Operating parameters (trifluoroacetic acid concentration, flow rate, and gradient slope) were evaluated using different enzymatic digests of horse cytochrome c and bovine serum albumin. Peptides ranging in size from five amino acids to 68 kDa could be separated on the propylsilyl column in a single chromatographic run. The cyanopropylsilyl column is suitable as a supplement to the use of the large-pore column for medium size (5-20 amino acids) peptides. The chromatographic supports and conditions presented here offer a simple, sensitive, and rapid separation system for a wide size range of peptides and proteins. They extend the versatility of separation methodology for these molecules.     

Factors influencing chromatography of proteins on short alkylsilane-bonded large pore-size silicas

The separation of proteins by high-performance gradient chromatography on short alkylchain bonded silica was studied with respect to pore size, mobile-phase composition, and temperature. Selectivity could be increased in particular cases by varying temperature or eluate compositon. Recovery of late-eluting, hydrophobic proteins was found to increase with flow rate and gradient slope. Recovery was also shown to be dependent on eluate composition—decreasing with added salt. The applicability of reverse-phase chromatography to proteins as large as 150 kD was demonstrated by the separation of monoclonal immunoglobulin.     

Reversible labeling of cysteine-containing peptides allows their specific chromatographic isolation for non-gel proteome studies

We report upon a novel procedure to specifically isolate cysteine-containing peptides from a complex peptide mixture. Cysteines are converted to hydrophobic residues by mixed disulfide formation with Ellman's reagent. Proteins are subsequently digested with trypsin and the generated peptide mixture is a first time fractionated by reverse-phase high-performance liquid chromatography. Cysteinyl-peptides are isolated out of each primary fraction by a reduction step followed by a secondary peptide separation on the same column, performed under identical conditions as for the primary separation. The reducing agent removes the covalently attached group from the cysteine side chain, making cysteine-peptides more hydrophilic and, thereby, such peptides can be specifically collected during the secondary separation and are finally used to identify their precursor proteins using automated liquid chromatography tandem mass spectrometry. We show that this procedure efficiently isolates cysteine-peptides, making the sample mixture less complex for further analysis. This method was applied for the analysis of the proteomes of human platelets and enriched human plasma. In both proteomes, a significant number of low abundance proteins were identified next to extremely abundant ones. A dynamic range for protein identification spanning 4-5 orders of magnitude is demonstrated.     

Use of reverse-phase high-performance liquid chromatography in structural studies of neurophysins,photolabeled derivatives,and biosynthetic precursors

The neurophysins are a class of hypothalamo-neurohypophyseal proteins that function as carriers of the neuropeptide hormones oxytocin and vasopressin. Currently, we are using reverse-phase high-performance liquid chromatography for structural characterization of the neurophysins, their chemically modified derivatives, and biosynthetic precursors. A cyanopropylsilyl (Zorbax CN) matrix has been found to be efficient and convenient for separation of major tryptic peptides of performic acid, oxidized or reduced, and alkylated neurophysins. Using this peptide mapping system we have studied the site of modification of a photoaffinitylabeled derivative of bovine neurophysin II by separation and identification of covalently modified peptides. In addition, this system has been used for mapping subfemtomole amounts of radioactively labeled biosynthetic precursors of the neurophysins. This procedure has allowed identification of neurophysin sequences within both pre-pro-neurophysins produced by in vitro translation and rat pro-neurophysins produced by in vivo pulse labeling.     

Partial isotope fractionation during high-performance liquid chromatography of deuterium-labelled internal standards in plant hormone analysis: A cautionary note

Deuterium-labelled indole-3-acetic acid, abscisic acid and phthalimido-1-aminocyclopropane-1-carboxylic acid were found to separate from the unlabelled compounds on reverse-phase high-performance liquid chromatography (HPLC). A similar separation was found for the methyl esters of these compounds on normal-phase HPLC. Such separations may lead to substantial errors when these compounds are used as internal standards for quantitation by gas chromatography-mass spectrometry/selective ion detection, unless the complete chromatographic peaks are collected.Abbreviations ABA abscisic acid - ACC 1-aminocyclopropane-1-carboxylic acid - IAA indole-3-acetic acid - GC-MS gas chromatography-mass spectrometry - HPLC high-performance liquid chromatography - Pht-ACC phthalimido-ACC - SIM selected ion monitoring     

Determination of low levels of 4-fluorophenylalanine incorporation into proteins

This paper describes a rapid method for the determination of low levels of 4-fluorophenylalanine biosynthetically incorporated into proteins. Precolumn derivatization with phenylisothiocyanate followed by reverse-phase high-performance liquid chromatography enables determination of the amino acid composition of the protein as well as accurate detection of a 1-3% substitution of phenylalanine by fluorophenylalanine.     

Rapid purification of bacterially expressed fusion proteins by high-performance liquid chromatography methods

Recent developments in recombinant DNA technology allow the high-level expression in bacteria of substantial amounts of viral and eukaryotic proteins whose genes have been cloned into plasmids. The present study reports two high-performance liquid chromatography (HPLC) methods for the rapid purification to apparent homogeneity of these bacterially expressed proteins. The two methods are anion exchange HPLC in the presence of 7 M Urea and reverse-phase HPLC of protein solubilized by 7.0 M guanidine hydrochloride. The two methods have been used successfully to purify fusion products of the v-myb oncogene and fusion proteins from HTLV-I Px and transmembrane regions and should be of general utility for purification of other bacterially produced proteins.     

A simple quantitative method for the determination of 3-fluorotyrosine substitution in proteins

A rapid quantitative method is described for determining 3-fluorotyrosine incorporation into proteins. Derivatives of tyrosine and 3-fluorotyrosine with o-phthalaldehyde are well separated from one another by a reverse-phase high-performance liquid chromatography system used for routine analyses of o-phthalaldehyde-amino acid derivatives. Since both amino acids are well resolved from all other derivatized amino acids, the method is useful for amino acid analyses of proteins. Determination of the fluorotyrosine content of proteins by this method involves a single separation step, is reproducible, and requires no corrections for stability or yield. Further, the o-phthalaldehyde derivatives of 5-fluorotryptophan, 2-fluorophenylalanine, 3-fluorophenylalanine, and 4-fluorophenylalanine can also be resolved. The method may be generally applicable to fluorinated aromatic amino acid-labeled proteins that are studied structurally and dynamically by nuclear magnetic resonance.     

Isolation and identification of rutin as the major mutagen of red wine

The polyphenolics of a red wine were concentrated by salt-induced phase separation into acetone-alcohol and fractionated by Sephadex LH-20 and multi-layer counter-current chromatography. The mutagenicity of each fraction was evaluated by the Salmonella mutagenesis assay. The mutagen of red wine required activation by both rat-liver microsomal enzymes and human-fecal enzymes (fecalase). The mutagenic component of red wine was purified to homogeneity by reverse-phase high-performance liquid chromatography (RPHPLC) on Lichrosorb C18 and was identified as rutin by UV spectrometry, co-chromatography with authentic standard on RPHPLC and gas-liquid chromatography/mass spectrometry.