Pmol Level Research Articles

Structural analysis of proteins by capillary HPLC electrospray tandem mass spectrometry

Capillary high performance liquid chromatography (HPLC) with on-line ultraviolet detection was coupled with electrospray tandem mass spectrometry. Protein tryptic digests were analyzed at sample levels as low as 800 fmol. Collision-activated dissociation was used to obtain amino acid sequence information on peptides at the 5 pmol level. Protein spotted on to nitrocellulose membrance was digested with trypsin and the resultant peptide mixture was analyzed by capillary HPLC—MS. A deoxynucleotide binding protein isolated by affinity chromatography and sodium dodecylsulfate-polyacrylamide gel electrophoresis was digested in situ and analyzed using this system. The methodology presented in this paper offers a high sensitivity, high throughput strategy for analyzing the primary structure of proteins.

Microsequence analysis of peptides and proteins: IX. An improved, compact, automated instrument

We describe the construction of an improved, compact protein sequencer with a vertical flow path and continuous flow reactor (CFR). Unique features include a hexagonal valve for six fluid inputs to the CFR, which connects vertically to a transfer valve that allows sample, reagent, and solvent input to a conversion flask (CF). The simplified CF contains only two inputs at the top, one for sample, reagent, and solvent input, and the other a vent. The CF drains from the bottom, connecting to a switching valve which allows either delivery to waste or to an on-line HPLC for the analysis of phenylthiohydantoin amino acid derivatives. Approximately 90% of the sample is analyzed by use of a sonic flow detector. The overall vertical flow path of the sequencer is about 16 cm. The size of the instrument (25 w × 38 h × 44 d cm) is smaller than that of commercially available sequencers or HPLC systems. The performance of the instrument includes reduced background peaks and high-sensitivity sequence analysis at the 5–10 pmol level. The simplified sequencer is more economical and portable than conventional sequencers.

Characterization of O-linked oligosaccharide biosynthesis in cultured cells using paranitrophenyl α-D-GaINAc as an acceptor

Aryl-N-acetyl-alpha-galactosaminides (aryl-GalNAc) are acceptor substrates for UDP-Gal:alpha-GalNAc beta 1-3 galactosyltransferase and, in vivo, aryl-GalNAc have been shown to inhibit O-linked oligosaccharide biosynthesis (Kuan et al., J. Biol. Chem. 264, 19271, 1989). Since aryl-GalNAc, appears to enter viable cells and serve as an acceptor for O-glycosylation enzymes, the recovery and characterization of the aryl-oligosaccharides from cell culture medium may reflect cellular pattems of O-glycosylation. To pursue this possibility, the following paranitrophenyl-linked oligosaccharide standards were enzymatically synthesized and characterized by 1H-NMR: Gal beta 1-3(GlcNAc beta 1-6)Gal-NAc alpha-pNp; Gal beta 1-3(Gal beta 1-4GlcNAc beta 1-6)GalNAc alpha-pNp; SA alpha 2-3Gal beta 1-3(SA alpha 2-3Gal beta 1-4GlcNAc,beta 1-6)GalNAc alpha-pNp; SA alpha 2-3Gal beta 1-3GalNAc alpha-pNp. As a model system, MDAY-D2 lymphoid tumour cells were cultured for various periods in medium containing 2 mM GalNAc alpha-pNp. The secreted aryl-oligosaccharides were separated by Biogel P2 chromatography and DEAE HPLC, followed by further fractionation of the disialyl oligosaccharides on an Ultrahydrogel HPLC column. Absorbance of the paranitrophenyl aryl constituent at 303 nm allowed detection at the 10 pmol level and provided a relatively specific means of following the oligosaccharides. MDAY-D2 cells produced disialylated aryl-oligosaccharides at a rate of 20 pmol/h/10(6) cells with a half-time of transit to the cell surface of 13.6 min, a rate consistent with their movement from the Golgi to the cell surface by bulk flow.(ABSTRACT TRUNCATED AT 250 WORDS)

Reliable High-Performance Liquid Chromatographic Determination for Membrane-Bound Enzymes; Applications onto Guanylate Cyclase and 5′-Nucleotidase

Abstract A convenient, simple and reliable HPLC assay method for membrane-bound enzymes, such as guanylate Cyclase and 5′- nucleotidase, were developed. The membrane enzymes of guanylate cyclase had been assayed by determination of the product cyclic GMP by radioimmunoassay after column chromatographical separation of cyclic GMP, and 5′-nucleotidase had been assayed by colorimetrical phosphate assay method. The use of radioisotope is dangerous and requires a special laboratory, and photometrical assay method on membrane enzymes is difficult to be carried out reliably because membrane samples exhibit turbidity. RP-HPLC assay method for guanylate cyclase directly measures the product of cyclic GMP fluorimetrically, which afford a sensitive detection for cyclic GMP at pmol level. RP-HPLC assay method for 5′-nuc1eotidase also directly the product of aderosine by UV detection at 260 nm. Reliable stoichiometry was observed in both of enzyme assay, i. e., decrease in substrate of GTP and increase in product of cyclic GMP for guanylate cyclase, decrease in substrate of AMP and increase in product of adenine for 5′-nucleotidase. These assay method are successfully applied onto membrane samples and also onto serum samples.

Examination of automated polypeptide sequencing using standard phenyl isothiocyanate reagent and subpicomole high-performance liquid chromatographic analysis

The feasibility of accurate protein sequencing at the subpicomole level, using automated Edman chemistry and “on-line” HPLC analysis, was studied. Several modifications of the standard system were first introduced. A larger portion of the phenylthiohydantoin amino acids (70%) is analyzed. Dissolution in 10% acetonitrile is improved by short periodic bursts of argon. Losses on the column of subpicomole amounts of analytes, in the presence and absence of scavengers, were quantitated; they are related to destruction rather than to unspecific sticking to the stationary phase. Baseline drift, for a large part caused by the presence of ultraviolet absorbing N,N-dimethylphenylthiourea in solvent B, is completely eliminated by the addition of a twofold molar excess of tryptophan to solvent A. This allows real time recording of the 269-nm absorption detector signal at 0.0005 absorption unit full scale. The combined modifications result in an eightfold increase in sensitivity over standard methods. Sequence calling at the 2 to 10 pmol level, through visual inspection of chromatograms, becomes increasingly simple this way. Once the sequenceable signal drops below the 1 pmol level in the course of a run, meticulous comparison and matching of the preliminary calls with a spreadsheet of peak integration data are necessary for accurate assignments. Reliable sequencing, with signals at the subpicomole level, is now feasible for stretches of over 10 residues. Contaminating amino acids and polypeptides and incompletely removed reaction by-products constitute a major problem for analysis at this level. Future limits to sensitivity of Edman sequencing will primarily depend on improved micropreparations of proteins in cleaner environments, higher purity reagents and solvents, instrument miniaturization, and solid-phase techniques.

Tamdem quadrupole fourier transform mass spectrometry

Tandem quadrupole Fourier transform mass spectrometry is shown to be a practical technique for the determination of molecular weights. Samples in the mass range up to 6000 daltons can be routinely analyzed at the 0.1–1.0 pmol level. Ions indicative of molecular weight are generated by particle bombardment, transmitted to and trapped inside a superconducting magnetic field and mass analyzed. Additional structural information is obtained by exposing the trapped ions to photons generated by an argon fluoride excimer laser to effect laser photodissociation. Sequence analysis of oligopeptides fractionated by liquid chromatography is discussed. Application of the technique to the structural chacterization of proteins isolated by two-dimensional gel electrophoresis is also demonstrated.

Picomol Level Microanalysis of Biological Lipid Hydroperoxides

A chemiluminescence-high performance liquid chromatography (CL-HPLC) was developed [T. Miyazawa et al., Anal. Lett., 20, 915, (1987)] for pmol level micro-determination of lipid hydroperoxides present in biological samples such as human blood plasma, and its lipoprotein fractions, tissue organs of experimental animals, and cultured human fetal diploid cells [T. Miyazawa et al., Anal. Lett., 21, 1033, (1988); J. Biochem., 103, 744, (1988)]. The method involves separation of each lipid class from tissue total lipids with normal phase or reversed phase HPLC and post-column detection of hydroperoxide-dependent chemiluminescence that is generated by luminol oxidation during a reaction of lipid hydroperoxides with cytochrome c-heme. The high specificity for their hydroperoxide group enables an assay for a large range of hydroperoxide concentrations, with a detection limit of 10 pmol of phospholipid hydroperoxides, triglyceride hydroperoxides and tocopherol hydroperoxides, respectively.

Simple isocratic high-performance liquid chromatographic method for the separation of the six vitamers of vitamin B 6

A simple, sensitive and fast isocratic high-performance liquid chromatographic method has been developed for the separation of all six biologically active forms (vitamers) of vitamin B 6. The separation is accomplished using a strong cation-exchange column and a mobile phase of 0,1 M ammonium dihydrogenphosphate adjusted to pH 4.0. All six vitamers are separated within 20 min at a flow-rate of 1 ml/min. The concentration of the vitamers is determined with a fluorescence detector (excitation 290 nm; emission 389 nm). The within-run precision of the method expressed as the coefficient of variation is below 5% at the 25 pmol level. Pyridoxal 5′-phosphate can be determined using either pre- or post-column derivatization with sodium bisulfite. Application of the method to cell-free yeast culture media is presented.

Peptide sequencing of the chick oviduct progesterone receptor form B

The progesterone receptor form B has been isolated to apparent homogeneity from large scale preparations of laying hen oviduct cytosol. The quantities obtained were sufficient to monitor the separation of tryptic peptides on HPLC columns. Using a multi-dimensional microbore HPLC peptide purification protocol, several peptides were isolated in homogeneous form and sequenced up to 34 steps at the sub-40 pmol level using a gas phase sequenator. One of the peptides showed a striking homology with sequences of the putative steroid binding domain of the human glucocorticoid receptor; this region is also conserved in the human and chick estrogen receptor.

Automated pre-column derivatization of amino acids with o-phthalaldehyde by a reagent sandwiching technique

A totally automated method for the pre-column derivatization and analysis of the o-phthalaldehyde amino acids has been described. The derivatives are formed by combining the sample and reagent in a mixer in-line with the column and taking the mixture directly to the column after a precisely controlled reaction time. Reagent addition is by the use of a multi-port automated valve. Over-all precision of the method at the 50–100 pmol level is about 1%. The proposed method has several advantages over previously reported automated methods for this analysis: (a) The reaction time is precisely controlled and can be varied reproducibly if needed by altering the initial mobile phase flow-rate. (b) The use of the auto-sampler only for taking the sample should give better reproducibility than when used for taking sample and reagent. The full-loop mode of sampling can be used to improve sampling precision. (c) The auto-sampler trays are used for samples only, not for mixing vials and/or reagent vials. As many as 48 samples per day can potentially be analyzed. (d) There is no BASIC program needed to control the sampling, mixing, and injection of the samples. (e) Only the actual volume of sample analyzed is injected, undiluted with reagent and/or quenching solution. (f) The method is easier and less expensive to implement than systems employing robotics, although it is more expensive than procedures which use the auto-sampler for taking the sample and reagent due to the cost of the automated valve for reagent addition. (g) The derivatives are formed on-line and are not exposed to air or light. Whether this enhances the stability of the derivatives is open to speculation.

Modification of calf thymus DNA by methyl methanesulfonate. Quantitative determination of 7-methyldeoxyguanosine by mass spectrometry

Quantitation of 7-methyldeoxyguanosine (m 7dG) produced in the in vitro methyl methanesulfonate (MeMS) action on calf-thymus DNA is achieved by enzymatic degradation, liquid chromatographic separation and chemical ionization mass spectrometry. The total degree of methylation, measured by uptake of [ 14C] MeMS was 0.35%. Mass spectral analysis shows that m 7dG constitutes 84% of the total methylated product. It is also shown that tandem mass spectrometry allows detection of m 7dG, as the protonated base, down to 1 pmol level, suggesting that MS/MS analysis can be the method of choice in quantitation of the adducts of in vivo DNA modifications.

Sensitive amino acid analysis by reversed-phase high-performance liquid chromatography optimization of the o∼phthalaldehyde method for composition of picomole amounts of acid hydrolyzates

The o-phthalaldehyde method for pre-column derivatization of amino acids for compositional analysis has been optimized for quantitation at the low picomole level. As little as 20 pmol of peptide are hydrolyzed and 6 pmol can be derivatized for analysis. The method results in quantitative recovery of the amino acids and gives reliable quantitation even at the 6 pmol level. The study details the procedures involved in hydrolyzing small amounts of peptides. The effects of purity and stability of the o-phthalaldehyde reagent, pH, and composition of the mobile phase are discussed with a view to adaptation for use with various high-performance liquid chromatographic systems. Optimization of the derivatization conditions and strategies for lengthening column life are described. The relative performances of different reversed-phase columns from two different manufacturers are compared.

Use of narrow-bore high-performance liquid chromatography for microanalysis of protein structure

We report here systematic approaches to microanalysis of protein structures using narrow-bore high-performance liquid chromatography of protein, peptide and derivatized [phenylthiocarbamyl (PTC-) and phenylthiohydantoinyl (PTH-)] amino acids. The utilization of columns with small diameters (2 mm or less) has improved resolution and sensitivity and enabled protein structure analysis at the low pmol level. Preparative isolation of proteins and peptides of pmol quantity is achieved and separation and identification of PTC- and PTH-amino acids can be routinized at the low pmol to subpmol level. The use of diode-array detection enables simultaneous multiple-wavelength monitoring and spectral retreat, which greatly enhances flexibility and usefulness of the present methods. In combination with protein microsequencing techniques, these narrow-bore high-performance liquid chromatographic procedures can facilitate structural analysis of minutely available proteins of biochemical and physiological significance.

New sensitive high-performance liquid chromatographic method for p-tyrosine aminotransferase assay

A rapid, sensitive and specific procedure has been developed for the determination of p-tyrosine aminotransaminase activity. The assay is based on high-performance liquid chromatography ( HPLC) separation and electrochemical detection of the pyruvate product, which has been derivatized with hydroxylamine to form a stable oxime. Using this method the product at the low pmol level can be measured. A comparison of the kinetic parameters of the rat liver tyrosine aminotransferase and rat brain non-specific aspartate aminotransferase towards p-tyrosine has been made.

Specificity of binding of a strain of uropathogenic Escherichia coli to Gal alpha 1—-4Gal-containing glycosphingolipids.

A strain of Escherichia coli originally isolated from urine of a patient with acute pyelonephritis was studied in detail for binding to glycosphingolipids. Bacteria labeled metabolically with [14C]glucose were layered over a glycolipid chromatogram and bound bacteria were detected by autoradiography. The detection was down to a few ng of glycolipid (pmol level) under these assay conditions. At a test level of 500 ng all glycolipids (more than a dozen molecular species analyzed) with Gal alpha 1----4Gal as an internal or terminal part bound the bacteria strongly while glycolipids known to lack this sequence were negative. Conformational analysis using hard sphere calculations including the exo-anomeric effect showed a bend in the saccharide chain at this disaccharide with a largely hydrophobic surface of the convex side, probably being part of the binding epitope. Mixtures of glycolipids isolated from a human ureter scraping and from urinary sediments bound bacteria in the 2- to 7-sugar interval. Thus, this infectious strain of E. coli recognizes glycolipids being present in epithelial cells lining the urinary tract.

Ascorbic acid determination in serum and aqueous humour by high-performance liquid chromatography.

High-performance liquid chromatography on a Supelcosil LC-HN2 analytical column in weak anion exchange mode has allowed separation of ascorbic acid, in less than 5 min, from other interfering substances in serum and aqueous humour. UV monitoring at 254 nm enables ascorbic acid to be detected at 20 pmol level. A method for determination of ascorbic acid concentration in serum and aqueous humour is described, and values from 10 cataract patients are reported.

Analysis of phospho-amino acids and amino acid amides at the picomole level using 4′-dimethylaminoazobenzene-4-sulphonyl cholride

Two groups of biologically important unusual amino acids, (a) phospho-amino acids and (b) amino acid amides, can be quantatively analysed at the pmol level using the dimethylaminoazobenzenesulphonyl chloride precolumn derivatization technique. Conditons for the complete high-performance liquid chromatographic separation of both phospho-amino acids and amino acid amides from common amino acids are described.

Fluorometric determination of 5-hydroxyindole derivatives by high performance liquid chromatography with cobalt(II) chloride, sodium carbonate, and sodium hydroxide.

High-performance liquid chromatoghraphic determination of 5-hydroxyindole derivatives was established. The postcolumn derivatization with sodium carbonate-sodium hydroxide-cobalt(II) chloride reagent allowed the determination of pmol level of the substances. This technique was applied to the determination of 5-hydroxyindoleacetic acid in human urine.

Trace analysis of aldehydes by reversed-phase high-performance liquid chromatography and precolumn fluorigenic labeling with 5,5-dimethyl-1,3-cyclohexanedione

Dimedone (5,5-dimethyl-1,3-cyclohexanedione) is a highly specific and extremely sensitive reagent for the determination of aldehydes. We have adapted this reagent to the high-performance liquid chromatographic analysis of these compounds by precolumn derivatization. The reaction and chromatographic conditions were optimized. Using fluorimetric detection, the standard deviation at the 28 pmol level was about ±2% for most aldehydes. The detection limit was found to be about 30 fmol per injected aldehyde, but could be decreased by a factor of 100 by a single extraction step. Intercalibration with the standard 2,4-dinitrophenylhydrazine technique gave good agreement. Applications to various environmental and industrial samples are illustrated.

Micro-analysis of amino acids

An automated amino acid analyzer has been developed for the analysis of amino acids with the sensitivity at the 10–100 pmol level except for proline which requires >50 pmol. o-Phthalaldehyde, in the presence of 2-mercaptoethanol, is used for the fluorometric detection of amino groups (Roth, M. (1971) Anal. Chem. 43, 880–882). A post-column reaction of the amino acid with sodium hypochlorite (Bohlen, P. and Mellet, M. (1979) Anal. Biochem. 94, 313–321) gives oxidation products amenable to detection with o-phthalaldehyde. The instrument uses high-performance liquid chromatographic pumps capable of micro-flow rates with a minimum pulsation. The method is suitable for routine analyses of amino acids at picomole levels with reproducibility and accuracy comparable to the ninhydrin-based amino acid analysis.