scholarly journals Bioprocess monitoring: minimizing sample matrix effects for total protein quantification with bicinchoninic acid assay

2016 ◽  
Vol 43 (9) ◽  
pp. 1271-1280 ◽  
Author(s):  
Wieland N. Reichelt ◽  
Daniel Waldschitz ◽  
Christoph Herwig ◽  
Lukas Neutsch
Keyword(s):  
Total Protein ◽  
Matrix Effects ◽  
Protein Quantification ◽  
Bioprocess Monitoring ◽  
Sample Matrix ◽  
Bicinchoninic Acid

scholarly journals Heat and Pressure Treatments on Almond Protein Stability and Change in Immunoreactivity after Simulated Human Digestion

Nutrients ◽  
2018 ◽  
Vol 10 (11) ◽  
pp. 1679 ◽  
Author(s):  
Elisabetta De Angelis ◽  
Simona Bavaro ◽  
Graziana Forte ◽  
Rosa Pilolli ◽  
Linda Monaci
Keyword(s):  
Protein Stability ◽  
Total Protein ◽  
Protein Pattern ◽  
Protein Quantification ◽  
Protein Profiling ◽  
Enzyme Linked Immunosorbent Assay ◽  
Total Protein Content ◽  
Bioinformatic Tools ◽  
Sds Page ◽  
Allergenic Proteins

Almond is consumed worldwide and renowned as a valuable healthy food. Despite this, it is also a potent source of allergenic proteins that can trigger several mild to life-threatening immunoreactions. Food processing proved to alter biochemical characteristics of proteins, thus affecting the respective allergenicity. In this paper, we investigated the effect of autoclaving, preceded or not by a hydration step, on the biochemical and immunological properties of almond proteins. Any variation in the stability and immunoreactivity of almond proteins extracted from the treated materials were evaluated by total protein quantification, Enzyme Linked Immunosorbent Assay (ELISA), and protein profiling by electrophoresis-based separation (SDS-PAGE). The sole autoclaving applied was found to weakly affect almond protein stability, despite what was observed when hydration preceded autoclaving, which resulted in a loss of approximately 70% of total protein content compared to untreated samples, and a remarkable reduction of the final immunoreactivity. The final SDS-PAGE protein pattern recorded for hydrated and autoclaved almonds disclosed significant changes. In addition, the same samples were further submitted to human-simulated gastro-intestinal (GI) digestion to evaluate potential changes induced by these processing methods on allergen digestibility. Digestion products were identified by High Pressure Liquid Chromatography-High Resolution Tandem Mass Spectrometry (HPLC-HRMS/MS) analysis followed by software-based data mining, and complementary information was provided by analyzing the proteolytic fragments lower than 6 kDa in size. The autoclave-based treatment was found not to alter the allergen digestibility, whereas an increased susceptibility to proteolytic action of digestive enzymes was observed in almonds subjected to autoclaving of prehydrated almond kernels. Finally, the residual immunoreactivity of the GI-resistant peptides was in-silico investigated by bioinformatic tools. Results obtained confirm that by adopting both approaches, no epitopes associated with known allergens survived, thus demonstrating the potential effectiveness of these treatments to reduce almond allergenicity.

Comparison of Manual and Benzenesulfonyl Chloride-Semiautomated Thiochrome Methods for Determination of Thiamine in Foods

1981 ◽  
Vol 64 (3) ◽  
pp. 616-622
Author(s):  
Abdel-Gawad M Soliman
Keyword(s):  
Matrix Effects ◽  
Linear Regression Analysis ◽  
Food Products ◽  
Benzenesulfonyl Chloride ◽  
Sample Matrix ◽  
Sample Extract ◽  
Before And After ◽  
Aoac Method ◽  
Reproducible Method ◽  
Manual Methods

Abstract A semiautomated procedure was used to measure the fluorescence of sample extracts before and after the addition of benzenesulfonyl chloride (BSC). Addition of BSC inhibited thiochrome formation and provided a more representative blank based on the fluorescence of all the reactants except thiochrome. Thiamine standard was added to each sample extract so that thiamine concentration could be calculated after correcting for sample matrix effects on thiochrome fluorescence. Twenty food products were analyzed using this method, and the results were compared with those obtained using the manual AOAC method. The mean percent recoveries and standard deviations were 100.2 ± 5.3 and 101.1 ± 10.1 for the BSC-semiautomated and the AOAC manual methods, respectively. Replicate analyses using the BSC method gave an average coefficient of variation of 2.8%. Linear regression analysis showed that the BSC method gave higher values, with a mean increase of 14.8%, than those obtained using the manual method. Sixty-four percent of this difference was due to elimination of the column purification step and 36% was due to correcting for sample matrix effects on thiochrome and blank fluorescence. The BSC method provides a rapid, accurate, and reproducible method for thiamine assay in different food products, especially for those foods with low thiamine levels.

SIMS direct ion imaging in the mineralogical sciences

1996 ◽  
Vol 54 ◽  
pp. 698-699
Author(s):  
G. McMahon ◽  
L. J. Cabri
Keyword(s):  
Secondary Ion Mass Spectrometry ◽  
Matrix Effects ◽  
Matrix Material ◽  
High Sensitivity ◽  
Relative Sensitivity ◽  
Beam Current ◽  
Sensitivity Factor ◽  
Elemental Distribution ◽  
Sample Matrix ◽  
Secondary Ion

The use of secondary ion mass spectrometry (SIMS) has enjoyed increasing popularity in the mineralogical sciences owing to its high sensitivity to all elements in the periodic table with detection limits in the parts per million to parts per billion regime, coupled with the ability to display maps of elemental distribution at these detection levels with a spatial resolution of 1 μm. A description of the technique and its application to a wide variety of mineralogical problems has recently been reviewed.The drawback of SIMS is the rather complicated nature of quantification schemes necessitated by sample matrix effects, which refer to differences in the sensitivity for a given element in samples of different composition. These differences result from changes in the ionization efficiency and sputtering yield (sample matrix specific) as well as changes in secondary ion transmission and ion collection efficiencies (instrument specific). Therefore, the use of matrix-matched standards of known concentration is required to establish a calibration factor known as the relative sensitivity factor (RSF) which can be used to convert the experimentally measured secondary ion intensity into concentration values. Furthermore, the effect of changes in ion intensity caused by variations in primary beam current or analysis at different sample positions is removed by normalization to an ion species which represents the matrix material.

In-gel total protein quantification using a ninhydrin-based method

Amino Acids ◽  
2013 ◽  
Vol 45 (4) ◽  
pp. 1003-1013
Author(s):  
Sung Ung Kang ◽  
Seok Heo ◽  
Gert Lubec
Keyword(s):  
Total Protein ◽  
Protein Quantification

scholarly journals A Ponceau S Staining-Based Dot Blot Assay for Rapid Protein Quantification of Biological Samples

Gels ◽  
2022 ◽  
Vol 8 (1) ◽  
pp. 43
Author(s):  
Dario Lucas Helbing ◽  
Leopold Böhm ◽  
Nova Oraha ◽  
Leonie Karoline Stabenow ◽  
Yan Cui
Keyword(s):  
Biological Samples ◽  
Protein Quantification ◽  
Dot Blot ◽  
Tissue Samples ◽  
Bicinchoninic Acid ◽  
Wide Range ◽  
Dot Blot Assay ◽  
Ponceau S ◽  
Commercial Kits ◽  
Bca Assay

Despite the availability of a wide range of commercial kits, protein quantification is often unreliable, especially for tissue-derived samples, leading to uneven loading in subsequent experiments. Here we show that the widely used Bicinchoninic Acid (BCA) assay tends to underestimate protein concentrations of tissue samples. We present a Ponceau S staining-based dot-blot assay as an alternative for protein quantification. This method is simple, rapid, more reliable than the BCA assay, compatible with biological samples lysed in RIPA or 2x SDS gel-loading buffer, and also inexpensive.

Optimizing charge state distribution is a prerequisite for accurate protein biomarker quantification with LC-MS/MS, as illustrated by hepcidin measurement

2018 ◽  
Vol 56 (9) ◽  
pp. 1490-1497 ◽  
Author(s):  
Ellen M.H. Schmitz ◽  
Niels M. Leijten ◽  
Joost L.J. van Dongen ◽  
Maarten A.C. Broeren ◽  
Lech G. Milroy ◽  
...  
Keyword(s):  
Charge State ◽  
Matrix Effects ◽  
Solid Phase ◽  
Charge State Distribution ◽  
Protein Quantification ◽  
Deficiency Anemia ◽  
Clinical Consequence ◽  
State Distribution ◽  
Roc Curve Analysis ◽  
Anemia Of Chronic Disease

Abstract Background: Targeted quantification of protein biomarkers with liquid chromatography-tandem mass spectrometry (LC-MS/MS) has great potential, but is still in its infancy. Therefore, we elucidated the influence of charge state distribution and matrix effects on accurate quantification, illustrated by the peptide hormone hepcidin. Methods: An LC-MS/MS assay for hepcidin, developed based on existing literature, was improved by using 5 mM ammonium formate buffer as mobile phase A and as an elution solution for solid phase extraction (SPE) to optimize the charge state distribution. After extensive analytical validation, focusing on interference and matrix effects, the clinical consequence of this method adjustment was studied by performing receiving operating characteristic (ROC)-curve analysis in patients with iron deficiency anemia (IDA, n=44), anemia of chronic disease (ACD, n=42) and non-anemic patients (n=93). Results: By using a buffered solution during sample preparation and chromatography, the most abundant charge state was shifted from 4+ to 3+ and the charge state distribution was strongly stabilized. The matrix effects which occurred in the 4+ state were therefore avoided, eliminating bias in the low concentration range of hepcidin. Consequently, sensitivity, specificity and positive predictive value (PPV) for detection of IDA patients with the optimized assay (96%, 97%, 91%, respectively) were much better than for the original assay (73%, 70%, 44%, respectively). Conclusions: Fundamental improvements in LC-MS/MS assays greatly impact the accuracy of protein quantification. This is urgently required for improved diagnostic accuracy and clinical value, as illustrated by the validation of our hepcidin assay.

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