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.     

Rapid separation of acetylated oligosaccharides by reverse-phase high-pressure liquid chromatography.

Peracetylated saccharides were separated by chromatography on a reverse-phase support, eluting with mixtures of acetonitrile-water. Gradient elution for 2.5 h gave significant separations of all linear glucose oligomers containing up to 35 sugar residues. With isocratic elution retention was exponentially related to molecular mass and only slightly affected by linkage or anomeric configuration. The presence of glucosamine in various saccharides markedly reduced their retention.     

Separation of phycobiliprotein subunits by reverse-phase high-pressure liquid chromatography

Baseline separation of subunits of diverse phycobiliproteins was achieved by a reverse-phase HPLC gradient method with a C4 large-pore column and a solvent system consisting of 0.1% trifluoroacetic acid (TFA) in water and 0.1% TFA in 2:1 (v/v) acetonitrile:isopropanol. The procedure was successfully applied to cyanobacterial allophycocyanin and C-phycocyanins, an unusual phycocyanin from a marine cyanobacterium, red algal B- and R-phycoerythrins, and a cryptomonad phycoerythrin. The subunit sizes in these proteins range from about 7.5 to 30 kDa. Sample recovery was in excess of 85% in all cases. On-line spectroscopic analysis with a multiple diode array detector allowed determination of the type and number of bilins carried by each subunit.     

Amino acid analysis by dinitrophenylation and reverse-phase high-pressure liquid chromatography

The determination of amino acids has been achieved by reverse-phase high-pressure liquid chromatography of their dinitrophenyl derivatives. The methods developed permit the quantitation of all amino acids commonly encountered in a protein hydrolysate and the effect of various parameters on this separation was systematically evaluated. The procedure eliminates the need for specialized postcolumn equipment as employed in conventional amino acid analysis and can be obtained by a simple gradient high-pressure chromatograph. The sensitivity obtained is comparable to that available by methods in common usage, being able to determine amino acids quantitatively in the low picomole range.     

The analysis of nanogram levels of free amino acids by reverse-phase high-pressure liquid chromatography.

A simple method and apparatus are described for the efficient recovery of proteins from sodium dodecyl sulfate-polyacrylamide gel systems after electrophoretic resolution. This procedure provides for high yields of proteins which are free of sodium dodecyl sulfate and in certain cases, exhibit significant levels of biological activity.     

Analysis of Neisseria gonorrhoeae peptidoglycan by reverse-phase, high-pressure liquid chromatography

The muramidase digest of peptidoglycan from Neisseria gonorrhoeae was isolated and analyzed by the use of a reverse-phase, high-pressure liquid chromatography system. As was found previously in the case of Escherichia coli, gonococci peptidoglycan is also composed of a greater number of muropeptides than can be resolved with thin-layer chromatography systems. Preliminary classification of the muropeptide components into subclasses based on O-acetyl modification and degree of cross-linkage was achieved. Examination of a penicillin-susceptible strain and a highly resistant strain with two penicillin-binding protein alterations synthesized distinctly different peptidoglycan structures, as revealed by this technique.     

Resolution of some glycopeptides of hen ovalbumin by reverse-phase high-pressure liquid chromatography

Reverse-phase high-pressure liquid chromatography has been shown to be useful in fractionation of oligosaccharides containing N-acetyl amino sugars on the basis of stereochemical differences. Data in this paper show this same stereochemical selectivity can be obtained for glycopeptides. Using dilute aqueous phosphate for elution, three major high-mannose glycopeptides from ovalbumin can be separated on the analytical scale in a few minutes and the same method can also be used for preparation of the glycopeptides on the milligram scale. High-field proton NMR spectroscopy is used to identify two of the compounds as the mannose-5 and -6 glycopeptides (E3 and D3) and the third as the mannose-6 glycopeptide substituted by two N-acetyl glucosamine (GlcNAc) residues (C3B). The NMR spectra and analytical chromatograms of the glycopeptides show high purity and freedom from contaminants previously identified in ovalbumin glycopeptides.     

Fractionation of oligosaccharides containing N-acetyl amino sugars by reverse-phase high-pressure liquid chromatography

High-pressure liquid chromatography (hplc) of N-acetylglucosamine (GlcNAc) and galactosamine (GalNAc) containing carbohydrates was performed on several reverse-phase silica columns. Nanomolar level detection was accomplished using far uv-absorbance monitoring. Baseline separations of the α and β anomers of GlcNAc, chitobiose, chitotriose, and chitotetraose were observed with water elution of the reverse-phase column. With the addition of up to 3% acetonitrile to the eluting solvent, similar resolution of chitin oligomers up to a chain length of seven was observed. Anomerization of the residue could be followed by isolation of either anomeric peak with subsequent rechromatography. Reduction of chitotriose with borohydride yielded a single sharp peak with a retention volume similar to that of the reducing trisaccharide. Semipreparative reverse-phase hplc allowed for the separation and identification by ¹³C NMR of the GlcNAc-α-1→6 GlcNAc disaccharide from the β-1→6 isomer. Methyl glycosides of GalNAc and GlcNAc were shown to have retention times much longer than the free sugar.     

Primary structure of flavocytochrome b2 from baker's yeast. Purification by reverse-phase high-pressure liquid chromatography and sequencing of fragment alpha cyanogen bromide peptides

Reverse-phase high-pressure liquid chromatography has been used for the purification of some large cyanogen bromide peptides from flavocytochrome b2 fragment alpha. Acetonitrile gradients at acid and/or neutral pH using mu Bondapak C18 columns were useful for the smaller peptides (43 and 67 residues). The two larger ones, alpha CB1 and alpha CB2, could only be separated from each other by trifluoroacetic acid/1-propanol gradients on mu Bondapak-CN columns. The various systems tested are presented and compared. The elucidation of the amino acid sequence of alpha CB2 (95 residues), alpha CB3 (67 residues) and alpha CB4 (43 residues) is described. The fragments were digested with trypsin, chymotrypsin and Staphylococcus aureus V8 protease as necessary. Fragment alpha CB2 was also cleaved at the unique tryptophanyl bond with cyanogen bromide. Peptides were fractionated by Sephadex chromatography, thin-layer finger-printing and/or high-pressure liquid chromatography. Peptides were sequenced mostly in the liquid phase sequenator. The cyanogen bromide peptides could be ordered using information obtained previously, as well as additional data obtained in this work. Together with the previous elucidation of cytochrome b2 core sequence and of the hinge region [Guiard, B. and Lederer, F. (1976) Biochimie (Paris) 58, 305--316; Ghrir, R. and Lederer, F. (1981) Eur. J. Biochem. 120, 279--287], the present results enable us to present the complete sequence of fragment alpha (314 residues) with only three overlaps missing between cyanogen bromide peptides. Sequence comparisons with other known flavoproteins do not indicate any noticeable similarity. Structural predictions indicate an alteration of alpha helices and beta structure. The possibility that the non-heme-binding portion of fragment alpha could constitute a flavin-binding domain is discussed.     

Microsequence analysis of peptides and proteins : I. Preparation of samples by reverse-phase liquid chromatography

Reverse-phase supports for the separation of peptides and proteins are compared in two high-performance liquid chromatographic systems. One uses a trifluoroacetic acid-acetonitrile solvent system with a 206-nm detector, and the other uses pyridine-formate or pyridine-acetate and 1-propanol with a postcolumn fluorescence detector. Each system was examined with RP8, RP18, and alkylphenyl supports. In most applications, the trifluoroacetic acid-acetonitrile solvent system used in conjunction with an alkylphenyl column performed best. The use of this system for the preparation of low-microgram amounts of samples for microsequence analysis is illustrated.     

Identification of methionine-containing tryptic peptides of unstable beta-tubulin separated by reverse-phase high-performance liquid chromatography

Methods for examining altered regions in unstable mutant proteins are described. The strategy is illustrated using assembly defective Chinese hamster beta-tubulin subunits that are rapidly degraded in the cell. These unstable proteins are metabolically labeled to high specific activity and isolated as spots on two-dimensional gels. Conditions for the generation of tryptic peptides from gel pieces containing beta-tubulin and their subsequent resolution by HPLC have been worked out. Through a combination of dual labeling with various tritiated amino acids and [35S]methionine as well as partial sequence analysis, the identification of several HPLC peaks with the known sequence of beta-tubulin has been accomplished. This technique should greatly aid attempts to map the sites of mutational alterations in beta-tubulin polypeptides, and the general strategy should be readily applicable to other mutant proteins.     

Analysis by reverse-phase high-pressure liquid chromatography of phenylisothiocyanate-derivatized 1-aminocyclopropane-1-carboxylic acid in apple extracts

A rapid and sensitive method for the determination of 1-aminocyclopropane-1-carboxylic acid (ACC) in apple tissues is described. This method is based on the derivatization of ACC with phenylisothiocyanate, and the subsequent separation and quantification of the resulting phenylthiocarbamyl-ACC by reverse-phase high-pressure liquid chromatography. Phenylthiocarbamylation of ACC (and other amino acids) in apple extracts is complete within 20 min at room temperature. After removing solvents and reagent, the phenylthiocarbamyl derivatives are separated on an octadecyl reverse-phase column, eluted with a mixture of acetonitrile and sodium acetate buffer at pH 4.6, and monitored with a uv detector set at 254 nm. An analysis of apple extract can thus be achieved in 23 min and detect quantities as low as 1 pmol. Assays have been done to compare the efficiency of this method with that of a method using an ion-exchange amino acid analyzer and with that of Lizada and Yang's method [(1979), Anal. Biochem. 100, 140-145]. The latter method proved to yield markedly less accurate results than the other two, but the derivatization-HPLC method was preferred because of simplicity of operation and a better separation of ACC.     

Neurohypophyseal hormone-like peptides in the ovine pineal gland using reverse-phase liquid chromatography and radioimmunoassay

A method is described for the determination of the neurohormone contents of ovine pineal tissue by radioimmunoassay (RIA) after successive fractionation on gel filtration in formic acid and reverse-phase liquid chromatography (HPLC). This method gives a good resolution for the neurohormones vasopressin, vasotocin and oxytocin, without a significant interference of aspecific cross-reacting of peptides with the RIA. An acid extract from ovine pineal tissue was found to contain amounts of immunoreactive AVP- and OXT-like peptides, whereas an AVT-like peptide was not detectable over background levels after HPLC with post-column RIA. It is concluded from our results that an AVT-like peptide is not present in ovine pineal tissue, and the pineal AVP- and OXT-like peptides appeared to be associated to neurophysin molecules.     

Isolation of spectrin subunits by reverse-phase high-performance liquid chromatography.

We present a one-step uncomplicated method of separation of spectrin subunits. The method is based on reverse-phase HPLC employing an analytical C4 column. Reverse-phase HPLC combines the steps of dissociation and separation of spectrin subunits. The method can be applied to different spectrin isoforms. It can be used for analytical purposes, as well as for small-scale (<0.4 mg) isolation of spectrin subunits.     

Quantification of glutamine in proteins and peptides using enzymatic hydrolysis and reverse-phase high-performance liquid chromatography

An enzymatic method for hydrolyzing bovine milk proteins was developed. Purified milk proteins (alpha-lactalbumin, beta-lactoglobulin, and beta-casein) were hydrolyzed in 0.1 M Hepes buffer (pH 7.5) containing pronase E, aminopeptidase M, and prolidase at 37 degrees C for 20 h. Free glutamine and other amino acids were derivatized with phenylisothiocyanate and separated using a C18 Pico-Tag column. Amino acids were eluted from the column with an aqueous sodium acetate-acetonitrile gradient with detection at 254 nm. Glutamine recoveries from hydrolyzed alpha-lactalbumin, beta-lactoglobulin, and beta-casein were 78 +/- 4, 98 +/- 3, and 101 +/- 3% of the theoretical values, respectively. The recoveries of most amino acids were comparable with those obtained using acid hydrolysis, except for the recoveries of proline and acidic amino acids. These peptide bonds appeared to be resistant to enzymatic hydrolysis and also to inhibit the hydrolysis of adjacent amino acids. Free glutamine was found to be very stable (97% recovery) under the enzymatic hydrolysis conditions.     

Molecular weight separation of proteins and peptides with a new high-pressure liquid chromatography column.

A new high-pressure liquid molecular weight chromatography column was evaluated for its ability to separate proteins and peptides. The column was able to give a linear separation of compounds between 5,000–700,000 M_r. Chromatography of posterior pituitary extracts, tumor-associated fetal antigens, and estrogen receptors demonstrated the ability of the column to separate biological samples.     

Assay for ADP-ribosyl cyclase by reverse-phase high-performance liquid chromatography.

Cyclic ADP-ribose (cADPR), a natural metabolite of beta-NAD(+), is a second messenger for Ca(2+) signaling in T cells. As a tool for purification and identification of ADP-ribosyl cyclase(s) in T cells, a sensitive and specific enzymatic assay using 1,N(6)-etheno-NAD(+) as substrate was developed. A major problem-the sensitivity of 1,N(6)-etheno-cADPR toward the extraction medium perchloric acid-was solved by replacing the perchloric acid extraction procedure of nucleotides by a filtration step. Standard compounds for the HPLC analysis of ADP-ribosyl cyclases and NAD(+)-glycohydrolases, e.g., 1,N(6)-etheno-cADPR, 1,N(6)-etheno-ADPR, and 1,N(6)-etheno-AMP, were produced by ADP-ribosyl cyclase from Aplysia californica and dinucleotide pyrophosphatase. The assay was applied to subcellular fractions prepared from human Jurkat T cells. As a result ADP-ribosyl cyclase and NAD(+)-glycohydrolase activity could be detected and precisely quantified in different subcellular fractions indicating the presence of different isoenzymes in T cells.     

Desalting of nucleotides by reverse-phase high-performance liquid chromatography

Chromatography of AMP, NAD⁺, or NADH on a reverse-phase C₁₈ Porasil B column rapidly removes ammonium formate or potassium phosphate from 90% of the nucleotide. Earlier reports showed these salts could not be separated from nucleotides by conventional desalting using gel filtration.