Method For Determination Of Protein Research Articles

Protein quantification in ecological studies: A literature review and empirical comparisons of standard methodologies

Abstract Protein quantification is a routine procedure in ecological studies despite the inherent limitations of well‐acknowledged protein determination methods which have been largely overlooked by ecologists. Thus, we want to bridge this knowledge gap, in hopes of improving the way ecologists quantify proteins and interpret findings. We surveyed the ecological literature to determine how and why ecologists quantify proteins. To determine whether different quantification methods produce comparable results across taxa, and between populations of a single species, we estimated the protein content of eight phylogenetically diverse taxa, and of desert isopods fed different diets, using various derived protocols of the 'crude protein', Bradford and bicinchoninic acid approach (BCA) methods. We found that ecologists use many protein quantification procedures, often without reporting the crucial information needed to evaluate and repeat their methods. Our empirical work demonstrated that the three quantification methods examined, and their derived protocols, resulted in highly divergent protein estimations that were inconsistent in rank across taxa, preventing conversion between methods. We also found that different quantification methods yielded different answers to whether isopod protein content is affected by diet. We conclude that commonly used quantification techniques yield distinct protein estimations with varying precision, and no single method is likely to be more accurate than another across taxa which may lead to inconsistent results across taxa and between conspecifics. Inaccurate protein quantification may explain the observed mismatch between organismal N and protein that has plagued some recent studies and that contradicts the principles of ecological stoichiometry. We recommend using a single BCA protocol to reduce inconsistencies across studies, until the promising amino acid analysis becomes more affordable, accurate and accessible to ecologists. Until then, ecologists should consider the abovementioned drawbacks of protein quantification methods and interpret their results accordingly.

Effect of gluten on the quality of meat products

Article Details: Received: 2020-10-14 | Accepted: 2020-11-27 | Available online: 2021-01-31 https://doi.org/10.15414/afz.2021.24.mi-prap.80-84 The aim of the study was the evaluation of gluten quality of selected meat products. Gluten is a vegetable protein, which, due to its properties, is very often used in various sectors of the food industry and therefore in the production of meat products. Sensory evaluation, microbiological examination and gluten detection were performed in samples of sausages, Poultry frankfurter, Fine salami and Malokarpatská salami. The meat products came from different producers and were purchased from a retail network. Sensory quality was determined by assessing color, aroma, juiciness, fragility and taste using a 5-point rating scale. The microbiological examination included the determination of the total number of microorganisms, the number of psychrotrophic, coliform microorganisms and coagulase-positive staphylococci. The presence of gluten alone was detected using a commercially produced GlutenTox Home test kit as well as a PCR reaction. Individual testing of selected meat products showed minor differences in the overall quality of gluten free and gluten-free meat products. Keywords: meat products, salami, gluten, quality, microbiological examination References Abaffyová, Z. et al. (2015). Cereal grain a little different. Bratislava. Pediatria pre prax . 4, 140–146. Frič, P., Keil, R. (2011). Celiac disease for practice. Olomouc. Medicína pro praxi. 8, 354–359. Hulín, P., Dostálek, P., Hochel, I. (2008). Methods for determination of gluten proteins in food. Metódy stanovení lepkových bílkovin v potravinách. Chemické listy ,102(5), 327–337. https://www.academia.edu/18759054/Methods_for_determination_of_gluten_proteins_in_food Olexová, L. et al. (2006). Detection of gluten-containing cereals in flours and ‘‘gluten-free’’ bakery products by polymerase chain reaction. Food Control , 17(3), 234–237. https://doi.org/10.1016/j.foodcont.2004.10.009 STN EN ISO 4833-1. Microbiology of food chain. Horizontal method for the enumeration of microorganisms. Part 1: Colony count at 30 degrees C by the pour plate technique. (ISO 4833-1: 2013) STN EN ISO 6888-1. Microbiology of food and animal feeding stuffs. Horizontal method for the enumeration of coagulase-positive staphylococci ( Staphylococcus aureus and other species). Part 1: Technique using Baird-Parker agar medium (ISO 6888-1:1999) STN EN ISO 4832. Microbiology. General guidance for the enumeration of coliforms. Colony count technique Taylor, S. (1992). Chemistry and detection of food allergens. Food Technology , 46(5), 148–152. https://doi.org/10.1080/10408399609527761 Toldrá, F. (2002). Fermentation and starter cultures. Dry-cured meat products , Trumbull : Food & Nutrition Press Inc., 89–112. https://doi.org/10.1002/9780470385111 van Hengel, A. J. (2007). Food allergen detection methods and the challenge to protect food-allergic consumers. Analytical and Bioanalytical Chemistry , 389(1), 111–118. https://doi.org/10.1007/s00216-007-1353-5

Rapid and non-destructive determination of protein and starch content in agricultural powders using near-infrared and fluorescence spectroscopy, and data fusion

Triboelectrostatic separation is a promising technology for the fractionation of agricultural flours to produce protein- and starch-enriched fractions. Rapid determination of the protein and starch content of these powder fractions is required for process development, optimization and ultimately control. The suitability of near-infrared and fluorescence spectroscopy along with data fusion for the rapid determination of protein and starch content and the development of a robust multivariate regression model was the objective in this work. A total of 102 samples were collected following triboelectrostatic separation of bean flour and analyzed using NIR and fluorescence spectroscopy. The superior performance of partial least squares method for both protein and starch, confirmed that NIR spectroscopy is a reliable method for rapid determination of protein- and starch-rich powders. The NIR and fluorescence data fusion model performed with the highest accuracy to predict the protein content.

Thrombin: An Approach to Developing a Higher-Order Reference Material and Reference Measurement Procedure for Substance Identity, Amount, and Biological Activities.

Thrombin, the proteolytic enzyme that catalyzes the transformation of soluble fibrinogen to the polymerized fibrin clot, participates in multiple reactions in blood coagulation in addition to the clotting reaction. Although reference materials have existed for many years, structural characterization and measurement of biological activity have never been sufficient to permit claims of clear metrological traceability for the thrombin preparations. Our current state-of-the-art methods for protein characterization and determination of the catalytic properties of thrombin now make it practical to develop and characterize a metrologically acceptable reference material and reference measurement procedure for thrombin. Specifically, α-thrombin, the biologically produced protease formed during prothrombin activation, is readily available and has been extensively characterized. Dependences of thrombin proteolytic and peptide hydrolytic activities on a variety of substrates, pH, specific ions, and temperature are established, although variability remains for the kinetic parameters that describe thrombin enzymatic action. The roles of specific areas on the surface of the thrombin molecule (exosites) in substrate recognition and catalytic efficiency are described and characterized. It is opportune to develop reference materials of high metrological order and technical feasibility. In this article, we review the properties of α-thrombin important for its preparation and suggest an approach suitable for producing a reference material and a reference measurement procedure that is sensitive to thrombin’s catalytic competency on a variety of substrates.

Detecting Low Concentrations of Nitrogen-Based Adulterants in Whey Protein Powder Using Benchtop and Handheld NIR Spectrometers and the Feasibility of Scanning through Plastic Bag.

Nitrogen-rich adulterants in protein powders present sensitivity challenges to conventional combustion methods of protein determination which can be overcome by near Infrared spectroscopy (NIRS). NIRS is a rapid analytical method with high sensitivity and non-invasive advantages. This study developed robust models using benchtop and handheld spectrometers to predict low concentrations of urea, glycine, taurine, and melamine in whey protein powder (WPP). Effectiveness of scanning samples through optical glass and polyethylene bags was also tested for the handheld NIRS. WPP was adulterated up to six concentration levels from 0.5% to 3% w/w. The two spectrometers were used to obtain three datasets of 819 diffuse reflectance spectra each that were pretreated before linear discriminant analysis (LDA) and regression (PLSR). Pretreatment was effective and revealed important absorption bands that could be correlated with the chemical properties of the mixtures. Benchtop NIR spectrometer showed the best results in LDA and PLSR but handheld NIR spectrometers showed comparatively good results. There were high prediction accuracies and low errors attesting to the robustness of the developed PLSR models using independent test set validation. Both the plastic bag and optical glass gave good results with accuracies depending on the adulterant of interest and can be used for field applications.

Xenobiotica-metabolizing enzyme induction potential of chemicals in animal studies: NanoString nCounter gene expression and peptide group-specific immunoaffinity as accelerated and economical substitutions for enzyme activity determinations?

Xenobiotica-metabolizing enzyme (XME) induction is a relevant biological/biochemical process vital to understanding the toxicological profile of xenobiotics. Early recognition of XME induction potential of compounds under development is therefore important, yet its determination by traditional XME activity measurements is time consuming and cost intensive. A proof-of-principle study was therefore designed due to the advent of faster and less cost-intensive methods for determination of enzyme protein and transcript levels to determine whether two such methods may substitute for traditional measurement of XME activity determinations. The results of the study show that determination of enzyme protein levels by peptide group-specific immunoaffinity enrichment/MS and/or determination of gene expression by NanoString nCounter may serve as substitutes for traditional evaluation methodology and/or as an early predictor of potential changes in liver enzymes. In this study, changes of XME activity by the known standard XME inducers phenobarbital, beta-naphthoflavone and Aroclor 1254 were demonstrated by these two methods. To investigate the applicability of these methods to demonstrate XME-inducing activity of an unknown, TS was also examined and found to be an XME inducer. More specifically, TS was found to be a phenobarbital-type inducer (likely mediated by CAR rather than PXR as nuclear receptor), but not due to Ah receptor-mediated or antioxidant response element-mediated beta-naphthoflavone-type induction. The results for TS were confirmed via enzymatic activity measurements. The results of the present study demonstrate the potential applicability of NanoString nCounter mRNA quantitation and peptide group-specific immunoaffinity enrichment/MS protein quantitation for predicting compounds under development to be inducers of liver XME activity.

3D printed silicone platforms with laser-scattering protein detection under flow analysis conditions as a development of Silicone Optical Technology (SOT)

ABSTRACT In this work, the 3D printed silicone microfluidic systems with simultaneous laser-scattering detection for turbidimetric and nephelometric measurements are presented. As a model reaction, the Exton method for total protein determination has been chosen as the well-known analytical protocol. Presented research is a development of recently presented idea of Silicone Optical Technology (SOT). It can be considered as the next step for all-in-one 3D printed analytical system concept, which means that microfluidic platform as well as detection system parts can be constructed during one-step manufacturing process. During the investigations, three microfluidic platforms with different flow-mixing patterns were designed and optimized for flow rate and sample injection volume. The most promising system with so-called 3D mixing (mixing channel along three axes) was characterized by analytical parameters sufficient for total protein analysis: limit of detection of 3 mg/L, linearity in the range of 16 – 400 mg/L and 50 µL sample consumption. The analytical usefulness of developed microfluidic platform was proved by analysis of urine samples. Furthermore, the design and setting up of double detection system based on Arduino microcontroller have been discussed.

Effect of difluoroacetic acid and biological matrices on the development of a liquid chromatography–triple quadrupole mass spectrometry method for determination of intact growth factor proteins

In biological systems, variable protein expression is a crucial marker for numerous diseases, including cancer. The vast majority of liquid chromatography-triple quadrupole mass spectrometry-based quantitative protein assays use bottom-up methodologies, where proteins are subjected to proteolytic cleavage prior to analysis. Here, the effect of difluoroacetic acid and biological matrices on the developement of a multiple reaction monitoring based top-down reversed-phase liquid chromatography-triple quadrupole mass spectrometry method for analysis of cancer-related intact proteins was evaluated. Seven growth factors (5.5-26.5kDa; isoelectric points: 4.6-9.9) were analyzed on a wide-pore C4 column. The optimized method was performed at 30°C, using a 0.2mL/min flow rate, a 10 %B/min gradient slope, and 0.05% v/v difluoroacetic acid as a mobile phase modifier. The increase of mass spectrometry sensitivity due to the difluoroacetic acid (estimated limits of detection in biological matrices 1-500ng/mL) significantly varied for proteins with lower and higher charge state distributions. Matrix effects, as well as the specificity of the method were assessed for variable biological samples and pretreatment methods. This work demonstrates method development to improve the ability to target intact proteins directly by more affordable triple quadrupole mass spectrometry instrumentation, which could be beneficial in many application fields.

Methodological features of the spectrophotometric determination of proteins in biological fluids using reactions with brompyrogallol red

Determination of proteins in biological fluids is rather important for diagnostics in current clinical practice. The results of total protein determination depend on the amino-acid composition of the proteins present in the biological fluid. We discuss some aspects of the spectrophotometric determination of proteins in biological fluids, in particular, the methodological features of the technique based on the reaction of proteins with brompyrogallol red (BPGR). The most important advantage of BPGR in the determination of proteins in biological fluids is rather high and equal sensitivity of the dye to the proteins of albumin and globulin fractions, thus minimizing the errors attributed to the mismatch of the protein composition of the analyzed samples and calibration solutions used. The goal of the work is to study the impact of conditions and shelf life of the BPGR solution on the analytical properties of the solution in the spectrophotometric determination of proteins in biological fluids. Stability of the optical and analytical properties of BPGR solutions are studied using Fisher and Student criteria under conditions of different storage temperatures and nature of the stabilizer (ethanol or sodium benzoate) in the reagent solutions. Verification of the correctness of the total protein determination by the proposed method was carried out in spike tests. The introduced additives of standard solutions are prepared from the «Total protein» or «Albumin» calibrators. The developed method of the spectrophotometric determination of the mass concentration of proteins in the urine by the reaction with bromopyrogallol red was tested on real objects, metrologically certified and listed in the Federal register of certified measurement techniques. Analytical and metrological studies have shown that the developed method of protein determination with a reagent based on BPGR provides equal and high sensitivity of determination of albumin and globulin protein fractions in human biological fluids. To increase the shelf life of the reagent solution and preserve the analytical properties of the solution, we recommend to use ethanol as a stabilizer.

Comparative study of two methods for the determination of plasma proteins

Comparative study of two methods for the determination of plasma proteins

Milk Protein Analysis: an Overview of the Methods - Development and Application

Milk protein content is an important component of milk, especially from a nutritional point of view and also for payment purposes. The aim of work was to draw up an overview on reference and routine methods for protein determination. Reference methods perform accurate analyses comply according to the International Standard ISO whereas routine methods perform analyses using routine instrumental techniques for faster and cheaper results with acceptable accuracy and a large number of processed samples. In most of cases, using of routine indirect methods for milk protein analysis requires their specific calibrations according to biological kind of measured milk (cow’s, goat’s or sheep’s milk) or specific conditions of milk technology treatment. Also, the quality control measures have a significant role for result determination reliability.

Analiza proteina glijadina visoko-pritisnom tečnom hromatografijom

In order to develop a method for rapid, efficient and accurate detection and quantitative determination of gliadin proteins from wheat flour, the usage of different solvents and different separation conditions were investigated by reversed-phase high performance liquid chromatography (RP-HPLC). After albumin and globulins were removed from wheat flour (100 mg) with a salt solution, the gliadin extraction was achieved with ethanol, 1-propanol and isopropanol (50% v/v, 60% v/v and 70% v/v each). Then, the glutenin fractions were extracted using nitrogen and 50% (v/v) aqueous solution of 1-propanol containing Tris-HCl (0.05 mol/dm3 , pH = 7.5) at 60 °C, urea (2 mol/dm3 ) and dithioerythritol (1%). The separation and quantitative gliadin determination were carried out by RP-HPLC chromatography on C3 column maintained at different temperatures: 40 °C, 45 °C and 50 °C. In order to determine the absolute amounts of the protein type, gliadin protein standard was used. The obtained results in terms of repeatability, linearity and accuracy showed that RPHPLC chromatography could be used as a fast, relatively simple and reliable way to quantify all types of gluten proteins in wheat flour and can be applied to assess the quality of cereals as raw materials or in cereal products.

Determination of conjugated protein on nanoparticles by an adaptation of the Coomassie blue dye method

A new accurate spectrophotometric method for protein determination on nanoparticles is described. The method is based on the Coomassie blue dye that binds to the basic and aromatic amino acid residues of proteins, especially arginine and lysine. A known amount of reagent dye was mixed with a variety of protein-loaded nanoparticles. Thereafter the unconjugated reagent was mixed with excess protein (bovine serum albumin) and titrated. In this method, the reacted dye on the protein coating of nanoparticle is directly determined, in opposite to the conventional method, in which the conjugated protein is determined as the difference between the non-conjugated protein found in the supernatant after centrifugation, and the total amount of protein originally used. This method is able to measure amounts of coated protein lower than 1 ppm.•Simple and accurate method especially adapted for protein-coated nanoparticles.•The amino acid residues of protein in the nanoparticle surface react with Coomassie brilliant blue dye.•The unreacted dye is titrated with an excess of a standard protein.

Direct detection of cysteine peptidases for MALDI-TOF MS analysis using fluorogenic substrates

A method is described for the direct detection of unstable cysteine peptidase activity in polyacrylamide gels after native electrophoresis using new selective fluorogenic peptide substrates, pyroglutamyl-phenylalanyl-alanyl-4-amino-7-methylcoumaride (Glp-Phe-Ala-AMC) and pyroglutamyl-phenylalanyl-alanyl-4-amino-7-trifluoromethyl-coumaride (Glp-Phe-Ala-AFC). The detection limit of the model enzyme papain was 17 pmol (0.29 μg) for Glp-Phe-Ala-AMC and 43 pmol (0.74 μg) for Glp-Phe-Ala-AFC, with increased sensitivity and selectivity compared to the traditional method of protein determination with Coomassie G-250 staining or detection of activity using chromogenic substrates. Using this method, we easily identified the target digestive peptidases of Tenebrio molitor larvae by matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) analysis. The method offers simplicity, high sensitivity, and selectivity compared to traditional methods for improved identification of unstable cysteine peptidases in multi-component biological samples.

Rapid LC-MS Method for Accurate Molecular Weight Determination of Membrane and Hydrophobic Proteins.

Therapeutic target characterization involves many components, including accurate molecular weight (MW) determination. Knowledge of the accurate MW allows one to detect the presence of post-translational modifications, proteolytic cleavages, and importantly, if the correct construct has been generated and purified. Denaturing liquid chromatography-mass spectrometry (LC-MS) can be an attractive method for obtaining this information. However, membrane protein LC-MS methodology has remained relatively under-explored and under-incorporated in comparison to methods for soluble proteins. Here, systematic investigation of multiple gradients and column chemistries has led to the development of a 5 min denaturing LC-MS method for acquiring membrane protein accurate MW measurements. Conditions were interrogated with membrane proteins, such as GPCRs and ion channels, as well as bispecific antibody constructs of variable sizes with the aim to provide the community with rapid LC-MS methods necessary to obtain chromatographic and accurate MW measurements in a medium- to high-throughput manner. The 5 min method detailed has successfully produced MW measurements for hydrophobic proteins with a wide MW range (17.5 to 105.3 kDa) and provided evidence that some constructs indeed contain unexpected modifications or sequence clipping. This rapid LC-MS method is also capable of baseline separating formylated and nonformylated aquaporinZ membrane protein.

Reliability of Analytical Methods for Recombinant Human Insulin Quantification in the Bulk Crystals and in-Process Control

The aim of this study was to demonstrate the reliability of the protein determination methods used in the process of recombinant human insulin development before its scale up. The total protein content was measured by Bradford, molar extinction coefficient, and dry weight methods. The standards were analyzed using Mono-Q, Aquapore RP300, and Kromasil columns to calculate the concentrations of the proteins using the theoretical extinction coefficient and peak area. The following highly purified standards were used: batches B4-258 and QS009-010 of sulfonated fusion protein; batches B4-267, B4-268, RALF-018, HGUT-042, HGUT-043, and HGUT-045 of renatured fusion protein; the United States Pharmacopeia reference; and batch B4-253 of bulk insulin crystals. The results were analyzed using ANOVA or Student’s t-test at 95 % significance. The Bradford method showed up to 60 % variation for all evaluated standards, while the remaining two methods were consistent with each other. The chromatographic parameters were used to validate the analytical methods, and all results met the current guidelines of Brazilian regulatory agencies. The use of quality parameters and the statistical evaluation of the data demonstrated that analytical methods used in the in-process control are suitable for the intended purpose, which certifies the reliability of the generated data.

Automated NMR resonance assignments and structure determination using a minimal set of 4D spectra

Automated methods for NMR structure determination of proteins are continuously becoming more robust. However, current methods addressing larger, more complex targets rely on analyzing 6–10 complementary spectra, suggesting the need for alternative approaches. Here, we describe 4D-CHAINS/autoNOE-Rosetta, a complete pipeline for NOE-driven structure determination of medium- to larger-sized proteins. The 4D-CHAINS algorithm analyzes two 4D spectra recorded using a single, fully protonated protein sample in an iterative ansatz where common NOEs between different spin systems supplement conventional through-bond connectivities to establish assignments of sidechain and backbone resonances at high levels of completeness and with a minimum error rate. The 4D-CHAINS assignments are then used to guide automated assignment of long-range NOEs and structure refinement in autoNOE-Rosetta. Our results on four targets ranging in size from 15.5 to 27.3 kDa illustrate that the structures of proteins can be determined accurately and in an unsupervised manner in a matter of days.

Protein Determination-Method Matters.

The reported protein content of foods depends on the analytical method used for determination, making a direct comparison between studies difficult. The aim of this study was to examine and compare protein analytical methods. Some of these methods require extraction preceding analysis. The efficacy of protein extraction differs depending on food matrices and thus extraction yield was determined. Overall, most analytical methods overestimated the protein contents. The inaccuracies were linked to indirect measurements, i.e., nitrogen determination and subsequent conversion to protein, or interference from other chemical substances. Amino acid analysis is the only protein analysis method where interfering substances do not affect the results. Although there is potential for improvement in regards to the hydrolysis method, we recommend that this method should be the preferred for food protein determination.

The evidence of proteases in sprouted seeds and their application for animal protein digestion

It has been experimentally proven that germinated plant seeds, such as leek, red clover, broccoli, and others contain proteases, which are able to digest animal protein such as gelatin, bovine albumin, casein, and egg albumin. Preliminary tests were carried out with the use of a semi-qualitative gelatin test, which is often applied to prove the presence of fruit enzymes. Quantitative examinations were carried out with the use of a ninhydrin reaction for amino acid determination as well as the Bradford and Smith methods for protein determination. Respective calibration curves were obtained for glycine (amino acid analysis) as well as for egg albumin, bovine albumin, and gelatin (protein analysis), with a full statistical evaluation including Mandel and Lack-of-Fit tests to check for linearity. It has been proven that the selected germinated seeds containing proteases can be applied as an effective aid for animal protein digestion.

1H-detected MAS solid-state NMR experiments enable the simultaneous mapping of rigid and dynamic domains of membrane proteins

Magic angle spinning (MAS) solid-state NMR (ssNMR) spectroscopy is emerging as a unique method for the atomic resolution structure determination of native membrane proteins in lipid bilayers. Although 13C-detected ssNMR experiments continue to play a major role, recent technological developments have made it possible to carry out 1H-detected experiments, boosting both sensitivity and resolution. Here, we describe a new set of 1H-detected hybrid pulse sequences that combine through-bond and through-space correlation elements into single experiments, enabling the simultaneous detection of rigid and dynamic domains of membrane proteins. As proof-of-principle, we applied these new pulse sequences to the membrane protein phospholamban (PLN) reconstituted in lipid bilayers under moderate MAS conditions. The cross-polarization (CP) based elements enabled the detection of the relatively immobile residues of PLN in the transmembrane domain using through-space correlations; whereas the most dynamic region, which is in equilibrium between folded and unfolded states, was mapped by through-bond INEPT-based elements. These new 1H-detected experiments will enable one to detect not only the most populated (ground) states of biomacromolecules, but also sparsely populated high-energy (excited) states for a complete characterization of protein free energy landscapes.