A global drive to source additional and sustainable biomass for the production of protein has resulted in a renewed interest in the protein content of seaweeds. However, to determine accurately the ...potential of seaweeds as a source of protein requires reliable quantitative methods. This article systematically analysed the literature to assess the approaches and methods of protein determination and to provide an evidence-based conversion factor for nitrogen to protein that is specific to seaweeds. Almost 95 % of studies on seaweeds determined protein either by direct extraction procedures (42 % of all studies) or by applying an indirect nitrogen-to-protein conversion factor of 6.25 (52 % of all studies), with the latter as the most widely used method in the last 6 years. Meta-analysis of the true protein content, defined as the sum of the proteomic amino acids, demonstrated that direct extraction procedures underestimated protein content by 33 %, while the most commonly used indirect nitrogen-to-protein conversion factor of 6.25 over-estimated protein content by 43 %. We therefore determined whether a single nitrogen-to-protein conversion factor could be used for seaweeds and evaluated how robust this would be by analysing the variation in this factor for 103 species across 44 studies that span three phyla, multiple geographic regions and a range of nitrogen contents. An overall median nitrogen-to-protein conversion factor of 4.97 was established and an overall mean nitrogen-to-protein conversion factor of 4.76. We propose that the overall median value of 5 be used as the most accurate universal seaweed nitrogen-to-protein (SNP) conversion factor.
To date, there is no valid analytical method available to determine the total whey protein (TWP) content in cheese. Therefore, the aim of this study was to develop HPLC methods for whey protein ...determination in mature cheese. For that purpose, foil-ripened whey protein-enriched model cheese and traditional Edam-type cheese were produced and analyzed. Suitable protein extraction methods and subsequent analytical methods to determine the acid-soluble whey protein (ASWP) and TWP content in cheese were developed. To characterize the influence of proteolysis on native and denatured whey proteins, the ASWP and TWP contents were determined throughout the ripening process. Both chromatographic methods showed that the individual whey proteins (α-lactalbumin and β-lactoglobulin) were not degraded during ripening. However, the analyzed ASWP content increased by up to 25% throughout ripening. Compared to traditional Edam-type cheese, the β-lactoglobulin content in whey protein-enriched cheese (containing 30% high-heat milk) increased by a factor of 3.5. To evaluate the chromatographic results, two different calculation models were used to estimate a reference value for the TWP content in the manufactured cheese. Further studies are required to optimize the quantification of TWP content in hard, semihard, soft, and cream cheeses.
•Chromatographic methods to analyze whey proteins in Edam-type cheese were developed.•High proteolytic stability of α-lactalbumin and β-lactoglobulin in cheese was observed.•Whey-enriched cheese showed significant enrichment of β-lactoglobulin.•Calculation models to estimate the TWP content in model cheese were developed.
This perspective explores the feasibility of smart sampling with dried blood spots for the determination of proteins and peptides from human biomatrices using liquid chromatography coupled to mass ...spectrometry for clinical purposes. The focus is on innovative approaches to transform filter paper from a mere sample carrier to an active element in sample preparation, with the aim of reducing the need for extensive and intensive sample preparation in the conventional sense. Specifically, we discuss the use of modified cellulose to integrate sample preparation at an early stage of sample handling. The use of paper immobilized with either trypsin or monoclonal antibodies for protein digestion and affinity clean‐up is discussed as a potential benefit of starting sample preparation instantly at the moment of sampling to optimize time efficiency and enable faster analysis, diagnosis, and follow‐up of patients.
Soil extracts usually contain large quantities of dissolved humified organic material, typically reflected by high polyphenolic content. Since polyphenols seriously confound quantification of ...extracted protein, minimising this interference is important to ensure measurements are representative. Although the Bradford colorimetric assay is used routinely in soil science for rapid quantification protein in soil-extracts, it has several limitations. We therefore investigated an alternative colorimetric technique based on the Lowry assay (frequently used to measure protein and humic substances as distinct pools in microbial biofilms). The accuracies of both the Bradford assay and a modified Lowry microplate method were compared in factorial combination. Protein was quantified in soil-extracts (extracted with citrate), including standard additions of model protein (BSA) and polyphenol (Sigma H1675-2). Using the Lowry microplate assay described, no interfering effects of citrate were detected even with concentrations up to 5 times greater than are typically used to extract soil protein. Moreover, the Bradford assay was found to be highly susceptible to two simultaneous and confounding artefacts: 1) the colour development due to added protein was greatly inhibited by polyphenol concentration, and 2) substantial colour development was caused directly by the polyphenol addition. In contrast, the Lowry method enabled distinction between colour development from protein and non-protein origin, providing a more accurate quantitative analysis. These results suggest that the modified-Lowry method is a more suitable measure of extract protein (defined by standard equivalents) because it is less confounded by the high polyphenolic content which is so typical of soil extracts.
•Bradford and Lowry techniques for measuring protein in soil extracts were compared.•We found polyphenol content affected Bradford estimations substantially more than Lowry.•Citrate (often used to extract soil protein) was not found to be problematic for the Lowry assay.•Using the Lowry microplate method, analytical response due to polyphenol and protein is ascribed.
•Plant and animal protein enzymatic hydrolysates were evaluated for protein content.•Protein was determined using Kjeldahl, Dumas, colorimetric and far-UV methods.•Amino acid analysis was also used ...for protein determination.•Lowry, Dumas and amino acid analysis were the most positively correlated.
This work was carried out to identify accurate methods that could be recommended for the quantification of proteins in food protein hydrolysates. Following hydrolysis with 4% alcalase, the amount of protein in various hydrolysate samples was measured using seven different analytical methods. Although the data obtained using different methods varied, HPLC amino acid analysis with a Pico-Tag column indicated that the highest concentration of amino acids in the protein hydrolysates was present in the casein sample while the lowest amount of protein was found in the sample of hempseed studied. However, the amino acid analysis data was mostly positively correlated with the Dumas and Lowry methods. We conclude that where available, amino acid analysis provides the best estimate of protein content of hydrolysates but the Dumas and Lowry methods can also be recommended as alternatives.
•Milk adulteration by nitrogen-rich compounds is an important issue.•Standard (Kjeldahl) methods fail to identify adulteration by nitrogen compounds.•Classical spectrophotometric methods for protein ...are not sensitive to these compounds.•A combination of methods is able to recognize nitrogen addition to milk.•These methods can be used in combination to screen for milk adulteration.
The Kjeldahl method and four classic spectrophotometric methods (Biuret, Lowry, Bradford and Markwell) were applied to evaluate the protein content of samples of UHT whole milk deliberately adulterated with melamine, ammonium sulphate or urea, which can be used to defraud milk protein and whey contents. Compared with the Kjeldahl method, the response of the spectrophotometric methods was unaffected by the addition of the nitrogen compounds to milk or whey. The methods of Bradford and Markwell were most robust and did not exhibit interference subject to composition. However, the simultaneous interpretation of results obtained using these methods with those obtained using the Kjeldahl method indicated the addition of nitrogen-rich compounds to milk and/or whey. Therefore, this work suggests a combination of results of Kjeldahl and spectrophotometric methods should be used to screen for milk adulteration by these compounds.
Accurate determination of residual protein content in hydroxypropyl chitin (HPCH), a temperature-sensitive chitin derivative developed recently, is of great importance for biomedical applications. ...Coomassie brilliant blue (Bradford) method, Lowry method and Bicinchoninic acid (BCA) method were investigated to estimate the content of residual protein in HPCH. It was found that both Bradford and Lowry methods are not suitable for the protein assay in the HPCH sample. The BCA method could be used to determine protein content in HPCH solutions at low concentrations with good accuracy and precision and reasonable limit of detection and limit of quantitation for medical applications.
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•Bradford and Lowry methods not suitable for the protein assay in the HPCH sample.•BCA method used to analyze protein in HPCH solutions with good accuracy and precision.•Low CHPCH chosen in BCA assay with reasonable LOD and LOQ for medical applications.
The use of extracellular vesicles (EVs) generated by mesenchymal stem cells (MSCs) holds great promise as a novel therapeutic approach. Although their immunomodulatory and regeneration potential has ...been reported to be similar to that of MSCs, the use of MSC-derived EVs in clinical settings will require several problems to be resolved. It is necessary to develop a standardised and widely accepted isolation technology and to improve methods such as the quantification and characterisation of MSC-derived EVs. In this way, EV studies can be compared, the acquired knowledge can be safely transferred to clinical platforms and the clinical results can be evaluated appropriately. There are many procedures for the collection and analysis of vesicles derived from different cells; however, this review provides an overview of methods for the determination of the total protein amount, specific proteins, particle number, non-protein markers like lipids and RNA, microscopy and other methods focusing on MSC-derived EVs.
•A modified biuret method was developed for corn-based products in this study.•The proposed and Kjeldahl methods mostly showed good agreement.•The proposed method enhanced the accuracy of prediction ...on zein with BSA as standard.•The method improved the protein solubility for the thermal processed samples.
A modified biuret method suitable for protein determination of corn-based products was developed by introducing a combination of an alkaline reagent with sodium dodecyl sulfate (reagent A) and heat treatments. The method was tested on seven corn-based samples. The results showed mostly good agreement (P>0.05) as compared to the Kjeldahl values. The proposed method was found to enhance the accuracy of prediction on zein content using bovine serum albumin as standard. Reagent A and sample treatment were proved to effectively improve protein solubilization for the thermally-dried corn-based products, e.g. corn gluten meal. The absorbance was stable for at least 1-h. Moreover, the whole measurement of protein content only needs 15–20min more than the traditional biuret assay, and can be performed in batches. The findings suggest that the proposed method could be a timesaving alternative for routine protein analyses in corn processing factories.
The functional capacity of NK cells is dynamically tuned by integrated signals from inhibitory and activating cell surface receptors in a process termed NK cell education. However, the understanding ...of the cellular and molecular mechanisms behind this functional tuning is limited. In this study, we show that the expression of the adhesion molecule and activation receptor DNAX accessory molecule 1 (DNAM-1) correlates with the quantity and quality of the inhibitory input by HLA class I-specific killer cell Ig-like receptors and CD94/NKG2A as well as with the magnitude of functional responses. Upon target cell recognition, the conformational state of LFA-1 changed in educated NK cells, associated with rapid colocalization of both active LFA-1 and DNAM-1 at the immune synapse. Thus, the coordinated expression of LFA-1 and DNAM-1 is a central component of NK cell education and provides a potential mechanism for controlling cytotoxicity by functionally mature NK cells.
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