Introducing AAA-MS, a rapid and sensitive method for amino acid analysis using isotope dilution and high-resolution mass spectrometry

Accurate quantification of pure peptides and proteins is essential for biotechnology, clinical chemistry, proteomics, and systems biology. The reference method to quantify peptides and proteins is amino acid analysis (AAA). This consists of an acidic hydrolysis followed by chromatographic separation and spectrophotometric detection of amino acids. Although widely used, this method displays some limitations, in particular the need for large amounts of starting material. Driven by the need to quantify isotope-dilution standards used for absolute quantitative proteomics, particularly stable isotope-labeled (SIL) peptides and PSAQ proteins, we developed a new AAA assay (AAA-MS). This method requires neither derivatization nor chromatographic separation of amino acids. It is based on rapid microwave-assisted acidic hydrolysis followed by high-resolution mass spectrometry analysis of amino acids. Quantification is performed by comparing MS signals from labeled amino acids (SIL peptide- and PSAQ-derived) with those of unlabeled amino acids originating from co-hydrolyzed NIST standard reference materials. For both SIL peptides and PSAQ standards, AAA-MS quantification results were consistent with classical AAA measurements. Compared to AAA assay, AAA-MS was much faster and was 100-fold more sensitive for peptide and protein quantification. Finally, thanks to the development of a labeled protein standard, we also extended AAA-MS analysis to the quantification of unlabeled proteins.     

A rapid direct telomerase assay method using 96-well streptavidin plates

We have developed a high-throughput direct assay methodfor the assay of telomerase activity that improves on previous direct telomerase assays in two ways that allow larger numbers of samples to be conveniently processed: (i) 96-well streptavidin coated plates are used to bind and wash biotinylated primer extension products from the telomerase assay, as opposed to tubes containing streptavidin-coated magnetic beads; and (ii) storage phosphor-imagery is used instead of film autoradiography to detect telomerase products after being washed and released from the streptavidin-derivatized matrix. This method improves on previous direct assay methods using magnetic beads by allowing larger numbers of samples to be conveniently assayed. Also, the total activity of the radiolabeled nucleotides used in this procedure is significantly lower than that used in standard direct telomerase assays, lowering costs and exposure to radioactivity. We have validated the assay by repeating, in triplicate, the IC50 determination of rivanol, our previously identified telomerase inhibitor.     

Cloning and expression of a conjugated bile acid hydrolase gene from Lactobacillus plantarum by using a direct plate assay.

The conjugated bile acid hydrolase gene from the silage isolate Lactobacillus plantarum 80 was cloned and expressed in Escherichia coli MC1061. For the screening of this hydrolase gene within the gene bank, a direct plate assay developed by Dashkevicz and Feighner (M. P. Dashkevicz and S. D. Feighner, Appl. Environ. Microbiol. 53:331-336, 1989) was adapted to the growth requirements of E. coli. Because of hydrolysis and medium acidification, hydrolase-active colonies were surrounded with big halos of precipitated, free bile acids. This phenomenon was also obtained when the gene was cloned into a multicopy shuttle vector and subsequently reintroduced into the parental Lactobacillus strain. The cbh gene and surrounding regions were characterized by nucleotide sequence analysis. The deduced amino acid sequence was shown to have 52% similarity with a penicillin V amidase from Bacillus sphaericus. Preliminary characterization of the gene product showed that it is a cholylglycine hydrolase (EC 3.5.1.24) with only slight activity against taurine conjugates. The optimum pH was between 4.7 and 5.5. Optimum temperature ranged from 30 to 45 degrees C. Southern blot analysis indicated that the cloned gene has similarity with genomic DNA of bile acid hydrolase-active Lactobacillus spp. of intestinal origin.     

Cloning and expression of a conjugated bile acid hydrolase gene from Lactobacillus plantarum by using a direct plate assay.

The conjugated bile acid hydrolase gene from the silage isolate Lactobacillus plantarum 80 was cloned and expressed in Escherichia coli MC1061. For the screening of this hydrolase gene within the gene bank, a direct plate assay developed by Dashkevicz and Feighner (M. P. Dashkevicz and S. D. Feighner, Appl. Environ. Microbiol. 53:331-336, 1989) was adapted to the growth requirements of E. coli. Because of hydrolysis and medium acidification, hydrolase-active colonies were surrounded with big halos of precipitated, free bile acids. This phenomenon was also obtained when the gene was cloned into a multicopy shuttle vector and subsequently reintroduced into the parental Lactobacillus strain. The cbh gene and surrounding regions were characterized by nucleotide sequence analysis. The deduced amino acid sequence was shown to have 52% similarity with a penicillin V amidase from Bacillus sphaericus. Preliminary characterization of the gene product showed that it is a cholylglycine hydrolase (EC 3.5.1.24) with only slight activity against taurine conjugates. The optimum pH was between 4.7 and 5.5. Optimum temperature ranged from 30 to 45 degrees C. Southern blot analysis indicated that the cloned gene has similarity with genomic DNA of bile acid hydrolase-active Lactobacillus spp. of intestinal origin.     

A rapid method for purifying PCR products for direct sequence analysis

An HPLC approach for purification and sequencing of double-stranded DNA obtained directly from a PCR is described. This simple and reliable procedure has several advantages; the DNA fragment is rapidly eluted (less than 7 minutes), requires no organic cleanup, produces several hundred bases of sequence and is sensitive enough to obtain DNA sequence from a single 100-microliters PCR. This method is demonstrated by sequencing tumor necrosis factor alpha (TNF alpha) gene amplified from mouse tail DNA.     

A rapid and specific enzymatic method for the estimation of L-Arginine.

Arginine can be estimated spectrophotometrically by the oxidation of NADH using octopine dehydrogenase. The method is highly specific for this amino acid and is applicable to the estimation of arginine in the presence of mono- and disubstituted guanidino compounds.     

A rapid and sensitive method for HPLC cholesterol determination in bile.

A relatively little time consuming simple method based on the treatment of bile with cholesterol oxidase and subsequent high performance liquid chromatography measurement of the 3-ketocholesterol produced in order to determine the level of the cholesterol concentration is described. The method avoids bilirubin interferences, has high reproducibility and recovery assays give 100% values. It is highly sensitive and suitable for use in the determination of cholesterol concentrations in bile and other bilirubin containing biological fluids.     

Ursodeoxycholate: a common bile acid in gallbladder bile of Japanese subjects.

Bile acid composition was investigated in normal gallbladder-bile collected from the Japanese patients suffering from the diseases other than hepatobiliary tracts.In addition to cholate, chenodeoxycholate, deoxycholate and lithocholate, ursodeoxycholate was detected as a predominant bile acid in all cases tested and its quantity was higher than that of lithocholate in most cases.A simplified method has been developed for the quantitative determination of bile acids. They were derived to their methyl ester-trimethylsilyl ethers and determined by gas-liquid chromatography on a column of 3% poly-phenyldiethanol amine succinate-80-100 mesh Chromosorb WHP. Average recoveries of added amounts of standard bile acids were found to range from 97 to 100%.     

A new bile acid conjugate, ciliatocholic acid, from bovine gall bladder bile.

This study was carried out to investigate the occurrence of ciliatocholic acid in bovine gall bladder bile. Ciliatocholic acid was synthesized according to the method described by Bergstr?m and Norman for the synthesis of taurocholic acid. Elemental analysis, melting point, and the infrared spectrum of this substance were determined. An isolation procedure for ciliatocholic acid was established by stepwise elution with an HCl-ethanol solvent system using a Dowex-1 anion exchange resin column chromatographic technique. Ciliatocholic acid amounting to 158 mug (as ciliatine) per 100 ml of gall bladder bile was found in the fraction eluted with 0.01 N HCl in 50% ethanol. This coumpound was purified by preparative thin-layer chromatography and confirmed to be ciliatocholic acid from the hydrolytic stability, phosphorus determination, and chromatographic behavior. Thus, bovine gall bladder bile contains a small amount of ciliatocholic acid.     

Determination of fatty acid compositions of Bacillus cereus and related bacteria: a rapid gas chromatographic method using a glass capillary column.

A rapid gas chromatographic method for the determination of fatty acid compositions of Bacillus cereus and related bacteria is presented. By the use of a free fatty acid phase-coated glass capillary column, the complete separation of fatty acids, including the branched ones, was achieved. The method enables a more distinct differentiation of Bacillus species than can be obtained with packed columns.     

Determination of fatty acid compositions of Bacillus cereus and related bacteria: a rapid gas chromatographic method using a glass capillary column.

A rapid gas chromatographic method for the determination of fatty acid compositions of Bacillus cereus and related bacteria is presented. By the use of a free fatty acid phase-coated glass capillary column, the complete separation of fatty acids, including the branched ones, was achieved. The method enables a more distinct differentiation of Bacillus species than can be obtained with packed columns.     

Differential rapid analysis of ascorbic acid and ascorbic acid 2-sulfate by dinitrophenylhydrazine method

Ascorbic acid 2-sulfate (AAS) has recently been detected in animals (1,2), and the suggestion has been made that it is involved in some physiological functions (3–5).AAS has been determined by spectrophotometer measurements at 254 nm, ? = 17,000, after isolation from animal tissues (1,6,7). This procedure, however, is complicated and time consuming. Baker et al. (8) reported a rapid assay for AAS from biological samples based on a dinitrophenylhydrazine (DNPH) method which is a standard method for ascorbic acid (AA) determination in biological materials. In this method, AA is oxidized to the osazone by incubation with DNPH in a dilute sulfric acid solution. According to Baker et al. (8), AAS reacts with DNPH during a 3-hr incubation at 60°C but not during a 1-hr incubation at 37°C, and the difference in the reading at 540 nm between the two temperatures corresponds to AAS.This report is concerned with a more rapid and specific method with DNPH for differential measurement of AA and AAS, that is, the differential oxidation of AA and AAS with 2,6-dinitrophenolindophenol (2,6-Dye) and KBrO₃ and the determination of the osazone produced with the original method by Roe and Kuether (9).     

Detection of plant genes using a rapid, nonorganic DNA purification method

We have developed a simple procedure for the preparation of plant genomic DNA using FTA paper. Plant leaves were crushed against FTA paper, and the genomic DNA was purified using simple, nonorganic reagents. The 18S rRNA gene and the gene encoding the ribulose-1, 5-bisphosphate carboxylase/oxygenase large subunit (rbcL) from the chloroplast genome were detected by PCR amplification of DNA on FTA paper. DNA amplification was successful using extracts from 16 dicot and monocot plants. Studies of specific plant extracts revealed that extracts of leaf samples could be collected and stored at room temperature on FTA paper without a decrease in the DNA amplification success rate for more than a month. Both the 18S RNA gene and the rbcL gene were detected in the genomic DNA isolated from various soybean cultivars stored in this manner. Furthermore, by modestly increasing the number of cycles of DNA amplification, we were able to detect the uidA gene in transgenic tobacco and rice leaves as well as a single copy gene linked to the resistance gene of cyst nematode race 3 using genomic DNA isolated on FTA paper. These results demonstrate that genomic DNA isolated using FTA paper can be used for the detection of plant genes, from a wide range of plants with either high or low gene copy number and of either nuclear or cytoplasmic origin.     

Application of a rapid direct viable count method to deep-sea sediment bacteria

For the first time, a Live/Dead (L/D) Bacterial Viability Kit (BacLight ) protocol was adapted to marine sediments and applied to deep-sea sediment samples to assess the viability (based on membrane integrity) of benthic bacterial communities. Following a transect of nine stations in the Fram Strait (Arctic Ocean), we observed a decrease of both bacterial viability and abundance with increasing water (1250-5600 m) and sediment depth (0-5 cm). Percentage of viable (and thus potentially active) cells ranged between 20-60% within the first and 10-40% within the fifth centimetre of sediment throughout the transect, esterase activity estimations (FDA) similarly varied from highest (13.3+/-5.4 nmol cm(-3) h(-1)) to lowest values below detection limit down the sediment column. Allowing for different bottom depths and vertical sediment sections, bacterial viability was significantly correlated with FDA estimations (p<0.001), indicating that viability assessed by BacLight staining is a good indicator for bacterial activity in deep-sea sediments. Comparisons between total L/D and DAPI counts not only indicated a complete bacterial cell coverage, but a better ability of BacLight staining to detect cells under low activity conditions. Time course experiments confirmed the need of a rapid method for viability measurements of deep-sea sediment bacteria, since changes in pressure and temperature conditions caused a decrease in bacterial viability of up to 50% within the first 48 h after sample retrieval. The Bacterial Viability Kit proved to be easy to handle and to provide rapid and reliable information. It's application to deep-sea samples in absence of pressure-retaining gears is very promising, as short staining exposure time is assumed to lessen profound adverse effects on bacterial metabolism due to decompression.