Method For Quantitation Of Proteins Research Articles

18O‐labeling quantitative proteomics using an ion trap mass spectrometer

We describe a method for simultaneous identification and quantitation of proteins within complex mixtures. The method consists of 18O-labeling, a simple stable isotope-coding that requires merely enzymatic digestion in 18O-water, in combination with a capillary-liquid chromatography electrospray ion-trap mass spectrometer. In a separate experiment using the same sample and a spike test, we demonstrate that the difference ration was calculated accurately using the 18O-labeling method even if the protein was part of a complex mixture. Our data also suggest that the accuracy of the quantitation can be improved by averaging the difference ratios of several peptides. In comparing our method with the isotope-coded affinity tag (ICAT) method, we show that the 18O-labeling method has the advantages of better recovery and fewer isotope effects. Therefore, the 18O-labeling method is a powerful tool for large-scale proteomics applications.

Absolute quantitation of proteins by a combination of acid hydrolysis and matrix-assisted laser desorption/ionization mass spectrometry.

Quantitation by mass spectrometry is increasingly used to monitor protein levels in biological samples. Most of the current methods are based on the relative comparison of protein quantities but are not suited for the determination of the absolute amount of a given protein. Here we describe a method for the absolute quantitation of proteins that is based on amino acid analysis by matrix-assisted laser desorption/ionization mass spectrometry. Proteins are completely hydrolyzed by acid hydrolysis and then mixed with standards of isotopically labeled amino acids. For the presented study, lysine, leucine, and two different types of labeled arginine were examined as standards. Quantitation of proteins is then achieved by measuring the ratios of labeled and unlabeled amino acids. The method has a sensitivity down to the low-femtomole range and can be applied to quantitate proteins separated by gel electrophoresis. Furthermore, we demonstrate that a mixture of two proteins can be quantitated using two labeled amino acids simultaneously.

New Method for Protein Quantitation

New Method for Protein Quantitation

Protein and allergen assays for natural rubber latex products.

The major issues in the efforts to alleviate allergy to natural rubber latex (NRL) proteins are prevention of allergic reactions and decreasing future sensitization. An accurate estimate of the potential allergenicity of NRL products is essential as guidance to manufacturers and users. Development of a reliable in vitro method for the quantitation of NRL allergens has been impeded by (1) limited knowledge of the individual allergens among NRL proteins, (2) variations in the protein profiles among NRL products, and (3) heterogeneity of responses to NRL allergens among sensitized individuals. The current methods include measurement of either the total or the antigenic NRL proteins. Although these methods provide valuable information, all except one have not been validated or standardized. The lack of sensitivity and specificity of this standardized method and poor correlation with the allergenic protein levels have mandated the need for a better assay. In the past year, an immunologic method for the quantitation of all antigenic proteins has been developed as a national standard. The method provides a significant improvement in sensitivity and specificity of NRL protein measurement, but the question of relevance to allergen content remains open. In the last several years, the intensive research aimed at purifying and characterizing individual allergens among NRL proteins resulted in the identification of more than 10 major allergens. On the basis of this knowledge, present efforts are focused on the development of a method for quantitation of relevant allergenic NRL proteins.

Optimizing Reaction Conditions of the NanoOrange® Protein Quantitation Method for Use with Microplate-based Automation

Because of the rapidly expanding need for higher sample throughput in drug discovery, automation of corresponding biochemical analyses is desirable. In particular, automation of protein quantitation is crucial since its results are used extensively. Recently, a single-reagent fluorescent protein quantitation method (NanoOrange®) with attractive performance attributes has become available. While it can potentially be automated with liquid handling workstations, several of this method's reaction parameters need to be optimized. We studied the time and a temperature dependence of the NanoOrange protein quantitation reaction in ninety-six well black microplates using either a temperature-regulated hot block or a microwave oven as heat sources. Fluorescence of the NanoOrange reaction was quantified with a multimode microplate spectral scanner. Time-dependent heating profiles of filled microplates placed on hot blocks at fixed temperatures (45, 55, 65, 75, and 95°C) revealed temperature differences of 4–7°C cooler for the outside wells compared to the inner wells, however the maximum well temperature did not exceed 65°C. Similar time-temperature studies of microwave-heated microplates revealed an equilibrium temperature of 45–49°C that was 10–16°C lower than microplates that were block heated. The bovine serum albumin (BSA): NanoOrange standard curves created using a hot block increased in slope from 45°C to 55°C, but then remained constant from 65 to 85°C. Fluorescence of the BSA: NanoOrange standard curve created using a microwave oven was about half the magnitude of the hot block-derived curves, possible reflecting a lower energy transfer rate of the microwave oven. We conclude that the NanoOrange protein quantitation method can be automated if a microplate-compatible hot block with a 65–85°C surface can heat the microplate for minimum of 15 min prior to quantifying the reaction's fluorescence.

Optimizing Reaction Conditions of the NanoOrange® Protein Quantitation Method for Use With Microplate-based Automation

Because of the rapidly expanding need for higher sample throughput in drug discovery, automation of corresponding biochemical analyses is desirable. In particular, automation of protein quantitatio...

A thousand points of light: the application of fluorescence detection technologies to two-dimensional gel electrophoresis and proteomics.

As proteomics evolves into a high-throughput technology for the study of global protein regulation, new demands are continually being placed upon protein visualization and quantitation methods. Chief among these are increased detection sensitivity, broad linear dynamic range and compatibility with modern methods of microchemical analyses. The limitations of conventional protein staining techniques are increasingly being encountered as high sensitivity electrophoresis methods are interfaced with automated gel stainers, image analysis workstations, robotic spot excision instruments, protein digestion work stations, and mass spectrometers. Three approaches to fluorescence detection of proteins in two-dimensional (2-D) gels are currently practiced: covalent derivatization of proteins with fluorophores, intercalation of fluorophores into the sodium dodecyl sulfate (SDS) micelle, and direct electrostatic interaction with proteins by a Coomassie Brilliant Blue-type mechanism. This review discusses problems encountered in the analysis of proteins visualized with conventional stains and addresses advances in fluorescence protein detection, including immunoblotting, as well as the use of charge-coupled device (CCD) camera-based and laser-scanner-based image acquisition devices in proteomics.

A Rapid Method for Quantitation of Insoluble Polymeric Proteins in Flour

ABSTRACTThe baking properties of several genotypes of U.S. hard wheats grown in state nurseries for the Wheat Quality Council (WQC) were analyzed by the Hard Winter Wheat Quality Laboratory. Flours (250 mg) from each individual line and location were extracted three times with 50% 1‐ propanol (1 mL) for 5 min each. Samples were vortexed continually during extraction. This method was effective in removing most monomeric proteins. Negligible detectable protein was found in the third extract. Significant amounts of polymeric glutenin were also extracted. Pellets were oven‐dried (130°C) for 1 hr and analyzed for protein content using nitrogen combustion analysis. Protein remaining in the pellet consisted mainly of polymeric protein. The amount of gliadin and soluble polymeric protein could also be measured by separating the supernatant by size‐exclusion chromatography. Good correlations between dough strength parameters and amounts of pellet protein and the relative amount of pellet protein (pellet protein/flour protein) were found for all samples. This procedure was simple and rapid, with the potential of analyzing large numbers of samples per day with good reproducibility.

Developing a dye-based method of protein quantitation on nitrocellulose

time during the incubation periods for discussion of the blocking, washing, first antibody, conjugate, and detection steps. Moreover, this exercise allows for inclusion of the SDS-PAGE and transfer steps as separate laboratory exercises preceding the immunodetection phase. If protein gel electrophoresis and transfer equipment are not available, hybridoma supernatant can be directly added to nitrocellulose membrane strips using a dot-blot apparatus, with each protein-dot representing a different simulated target protein. This laboratory exercise is an effective procedure for demonstrating the theory and practice of the Western blot used in serodiagnosis.

Rapid and sensitive method of quantitation of bone gla protein mRNA using competitive polymerase chain reaction.

A method for sensitive quantitation of bone gla protein (BGP, osteocalcin) mRNA has been developed using competitive polymerase chain reaction after reverse transcription (competitive RT-PCR). The complementary DNA (cDNA) were transcribed from sample RNA was co-amplified in a PCR with a known amount of mutant BGP cDNA (competitor) using the identical oligonucleotide primers. The mutant cDNA with its unique restriction site allowed quantitation of sample and mutant PCR products after densitometric analysis of ethidium bromide-stained agarose gels. A linear relationship between initial sample BGP amount and the ratio of BGP to mutant BGP band intensity was obtained and used to make a standard curve to determine the initial BGP mRNA of unknown samples. These standard curves were made with known amounts of recombinant BGP cDNA. The competitive RT-PCR for BGP allows measurement of twofold differences in 1 and 2 micrograms total RNA and requires at least 10 times less sample RNA than usual Northern blotting. Moreover, heteroduplexes with one BGP strand and one mutant BGP strand formed as a result of high PCR cycles were quantifiable. This provided the advantages of rapid quantitation from ethidium bromide-stained gels without blotting, hybridization, or autoradiography. Multiple samples could be assayed for greater confidence in the results. The sensitivity, accuracy, and ease of the assay will facilitate analysis of BGP mRNA from a small amount of sample. The assay has been used to confirm the BGP mRNA changes with hormonal treatment in cultured cells and the age-related changes in whole tibia in vivo.

A Fast and Sensitive Method for Detection of Phospholipid-Binding Proteins on Nitrocellulose Membranes

We describe a sensitive new method for specific detection and quantitation of phospholipid-binding proteins using two purified lipocortins as model proteins. The method consists of blotting proteins to nitrocellulose membranes followed by incubation with radiolabeled phospholipids and autoradiographic detection of bound phospholipids. It allows specific detection of phospholipid binding proteins to a threshold of about 100 ng/spot and their quantitation up to several micrograms as well as rapid analysis of specific phospholipid binding properties of individual proteins. If performed as a dot-blotting technique, the method permits the simultaneous analysis of a large number of samples and the establishment of Ca2+ and phospholipid binding curves. If blotting is preceded by electrophoresis, the method also allows specific detection and quantitative estimation of phospholipid binding proteins in crude biological preparations and may thus be useful for monitoring and analysis of these proteins in various biological samples.

タンパク定量法による培養細胞増殖率測定法の検討

Two protein quantitation methods for measurement of cell growth in tissue culture were examined on their utility by using L 929 cell line. One was a modified Lowry method using a phenol reagent, and the other was the Dye binding assay method described by Bradford. The range of cell protein measurement was found to be enough to use and a little wider by the modified Lowry method than by the Dye binding assay method ; 5∿100μg by the former and 5∿60μg by the latter. The calculated factor to convert bovine serum albumin standard equivalent (μg) to cell count was 2.0×10^3 cells by the modified Lowry method, and 2.8×10^3 cells by the Dye binding assay method. Furthermore, those two methods were compared with other two methods to which protein quantitation technique was not applied ; one was nucleus counting through a microscope, and the other was a kind of colorimetric determination of the stained cell layer with Monocellater. Both of the protein quantitation methods were confirmed to give much less amount of scatter in measured values than nucleus counting or than the method with Monocellater.

Immobilization of binding proteins on nonporous supports

Four different nonporous particulate materials, nylon, polystyrene, soda-lime silicate glass, and fused silica glass, have been evaluated for their appropriateness as immobilization supports for immunoglobulins. A method of protein quantitation that is usually applied to solutions, the bicinchoninic acid (BCA) assay, was used successfully to directly measure ng amounts of protein immobilized on the supports. Two proteins, a monoclonal antibody to theophylline and the biotin binding protein avidin, were studied. Radioactive theophylline and radioactive biotin were used to measure the activity of the immobilized protein. Ligand binding capacity per mm2 of support was measured as a function of amount of protein immobilized. By measuring both the amount of protein immobilized and its ligand binding capacity, we have determined that antitheophylline antibody adsorbed on polystyrene balls loses almost 90% of its binding activity after 65 h, although little protein is lost from the balls over this time. Avidin retains nearly full activity for biotin on polystyrene. The binding activity of biotinyl-antibody conjugate immobilized on avidin-adsorbed polystyrene is stable, even when stored for over 22 wk. Antibody covalently immobilized on soda-lime silicate glass beads retains its binding activity over long-term storage, although only 0.1 mol of 3H-theophylline bind per mol of immobilized antibody. Using fused silica glass particles as the solid support, the same antibody binds approx 0.6 mol of ligand per mol of immobilized antibody protein. The structural "softness" of the immunoglobulin requires that interaction with the surface be prevented in order to maintain activity.

Application of fourth derivative absorption spectroscopy to protein quantitation during purification

A method for protein quantitation in the presence of nonprotein cellular components is described. The method is based on measurement of two tryptophanspecific signals in the fourth derivative of the protein's ultraviolet absorption spectrum, a peak at 283 nm and a trough at 288 nm. The amplitude between these two extremes is shown to vary linearly with protein concentration for bovine serum albumin and the outer membrane vesicles of Neissera meningitidis even when these protein solutions are supplemented with enough nucleic acid to completely obscure the parent absorption spectrum of the protein. The utility of this method as an in-process assay during isolation of a protein is demonstrated by comparing estimates of protein content from fourth derivative spectroscopy with those from the Lowry assay for samples at several steps along the isolation pathway for outer membrane vesicles of N. meningitidis. The advantages and limitations of the present method are discussed.

Simple method for quantitation of cell-bound protein A on Staphylococcus aureus cells by means of hemagglutination with sheep erythrocytes differentially sensitized with rabbit antibody and its clinical application.

A simple method for quantitation of cell-bound protein A (SpA) on organisms of Staphylococcus aureus was successfully devised by using hemagglutination between staphylococcal organisms and a series of sheep erythrocyte suspensions sensitized with different amounts of anti sheep erythrocyte rabbit antiserum. The validity of the principle and the reproducibility of the method presented here were precisely analyzed and the details of the method are presented. The hemagglutination was quantitatively inhibited both by normal rabbit serum and by soluble SpA. Using the method presented here, 376 strains of S. aureus freshly isolated from clinical materials were subjected to SpA quantitation in cell-bound form. According to the results, there was an unexpected distribution profile in the amounts of cell-bound SpA among the clinical isolates, which showed a two-peak pattern. A possible usefulness of the method presented here in clinical investigations is briefly discussed also.

Measurement of microgram quantities of protein by a generally applicable turbidimetric procedure

A modified turbidimetric method for protein determination which involves the use of trichloroacetic acid as the precipitating agent is described. Maximal turbidity develops in less than 30 min and is stable for at least 120 min. A linear relationship between turbidity at 340 nm and protein concentration is observed between 2 and 40 μg protein. Sodium dodecyl sulfate is added to avoid the interference by nonionic and cationic detergents and lipids and to decrease the procein-to-protein variation. The use of cetyltrimethyl ammonium bromide provides a two-step procedure to correct for the contribution of contaminating nucleic acid. Many compounds which interfere with other protein quantitation methods have no effect on this system. The interference of commonly used reagents as sucrose and urea can be easily corrected. This procedure compared favorably with the most widely used protein quantitation methods in simplicity, sensitivity, and specificity.

A sensitive and rapid immunochemical method for quantitation of proteins.

A sensitive and rapid ELISA for quantitation of seed globulins is described. This method employs conjugation of pigeon pea (Cajanus cajan) globulin antibodies and the enzyme peroxidase together with dextran. Using this conjugate, proteins as low as 0.1 ng were detected. Dextran conjugate has a ten-fold greater efficiency of quantitating pigeon pea globulins than the commercial goat anti-rabbit IgG conjugate, and is three-fold more efficient than pigeon pea globulin IgG peroxidase conjugate. The method can be conveniently adapted for quantitation of other proteins also.

Lowry protein determination by automated flow injection analysis for bovine serum albumin and hepatitis B surface antigen

The Lowry method for quantitation of protein was adapted to automated flow injection analysis. The procedure was developed using two different pure proteins: bovine serum albumin and hepatitis B surface antigen. The system was optimized for reagent concentration, pH, gain, temperature, sample volume, and output. The response of each protein was affected differently by temperature. The reaction slopes and absorbance values of the proteins were similar at 90°C to allow quantitation of hepatitis surface antigen against bovine serum albumin. Advantages of the automated flow injection analysis Lowry procedure include: rapid analyses (90 samples/h), small sample volume (30 μl, 100 μl), fast response (20 s), reproducibility (≤2% CV within an assay and 3 to 6% CV among assays), sensitivity (5 μg), and high correlation (99.8%) with manual assay. After a 30-min set-up period, the analyzer was available to assay protein on demand throughout the day, making it suitable for process and quality control testing.

Protein quantitation of as low as 10-ng/ml concentrations by competitive binding to polystyrene latexes

A protein quantitation method which offers protein detection as low as 10 ng protein/ml and accurate quantitation as low as 30–100 ng protein/ml, depending on the protein, has been designed. The assay, which is relatively quick and simple to perform, utilizes the strong, nonspecific adsorption of proteins onto polystyrene latexes. A competition is created between a marker enzyme and the analyte protein for a limited amount of latex surface area. Due to inactivation of the enzyme upon binding to a hydrophobic latex surface, measurement of enzyme activity allows determination of the bound/free enzyme ratio and thus the competing protein concentration. Considerations of sensitivity and simplicity are suggested to make this assay superior to others presently available.

A fluorescent method for the rapid staining and quantitation of proteins in sodium dodecyl sulfate‐polyacrylamide gels

AbstractFluorescence studies carried out in solution indicate that in the presence of the anionic detergent sodium dodecyl sulfate (SDS) proteins are able to bind the noncovalent dye 1‐anilinonaphthalene‐8‐sulfonate (ANS). The interaction of this hydrophobic dye with protein‐SDS complexes is probably responsible for the fluorescence of protein bands observed by UV‐transillumination of polyacrylamide gels stained with ANS. It is shown that the staining of SDS‐polyacrylamide gels with ANS after electrophoresis allows the detection and quantitative estimation of proteins and peptides having different properties. The electrophoretic patterns obtained using ANS are equivalent to those obtained using Coomassie Brilliant Blue R‐250. The fluorescent staining method described in this work is simpler and much more rapid than conventional methods using visible dyes.