Protein Quantification Method Research Articles

Peptides releasing study of serum apolipoprotein A-I and apolipoprotein B by ID-LC-MS

Objective To investigate the digestion kinetics of Apolipoprotein A-I and B by ID-LC-MS method for accurate quantification of proteins. Methods Methodological research. The target peptides of ApoA-I and B were determined. The ApoA-I and B from 5 human serum samples on market with levels from 0.90-2.54 g/L and 0.54-1.39 g/L separately, were measured in terms of target peptides by isotope dilution liquid chromatography mass spectrometry method. The releasing amount and rate of peptides were analyzed and plotted according to different time points. The correlation coefficient R2 was calculated among peptide releasing amount between samples. Results Most peptides reached their peaks within 4 hours. The peptides VQ, DY and VS from Apo A-I, TR and FP from Apo B were released relatively slowly. After getting to their peak stage, the ratio between TEV and SIL-TEV, AK and SIL-AK, VQ and SIL-VQ presented stable state. As for Apo A-I the correlations among peptides are high, from 0.904 to 0.999. Some peptides from Apo B show lower correlations, such as TG-SV with R2 0.543 (3 h). Conclusions Peptides from Apo A-I and Apo B present different releasing properties after trypsin digestion. Proper selection of representative peptides and enzymatic conditions can benefit accurate quantification of target proteins.(Chin J Lab Med, 2018, 41: 870-874) Key words: Apolipoprotein A-I; Apolipoproteins B; Kinetics; Chromatography, liquid; Tandem mass spectrometry; Isotope labeling

Development and comparative analysis of yeast protein extraction protocols for mass spectrometry

Mass spectrometry is the most used method for protein identification and quantification. Here we developed four protein extraction protocols precisely for mass spectrometry, and we compared with other ones already published. The best protocol developed by us consists on a simple extraction solution, a heat-shock step, and does not use protease inhibitor; moreover, it is the most efficient and uniform among replicates, besides to be safe, cheap and fast. That method also provided the highest number of proteins uniquely identified and allows finding a diversity of protein classes, which their absence is a problem to be avoided.

Undergraduate laboratory experiment on determination of total protein content in milk powder by moving reaction boundary titration.

Laboratory exercises focused on protein quantification are frequently conducted in traditional undergraduate biochemistry laboratory curriculum. The laboratory course described here is designed to provide students with experience in measurement of protein content in milk powder by moving reaction boundary titration (MRBT), a new rapid technique for total protein content determination in milk. In addition, this approach is weakly influenced by nonprotein nitrogen reagents such as melamine and urea. The course was done as three weekly laboratory exercises. First, students established a standard curve for milk protein concentration by MRBT method. Then, students investigated the influence of nonprotein nitrogen reagents on MRBT method. Finally, students made a comparison among three different protein quantification methods (MRBT, Biuret, and Kjeldahl method). From the experiments, students grasped the concept and advantages of MRBT and deepened the understanding of protein quantification. This course offer students the opportunity to be exposed to an advanced technique, which may have practical significance to their future study and work in the life science field. © 2018 International Union of Biochemistry and Molecular Biology, 46(6):644-651, 2018.

Optimization of sample preparation for transporter protein quantification in tissues by LC–MS/MS

BackgroundReproducible quantification of drug transporter protein expression in tissues is important for predicting transporter mediated drug disposition. Many mass-spectrometry based transporter protein quantification methods result in high variability of the estimated transporter quantities. Therefore, we aimed to evaluate and optimize mass spectrometry-based quantification method for drug transporter proteins in tissues. Materials and methodsPlasma membrane (PM) proteins from mouse tissues were isolated by applying three extraction protocols: commercial plasma membrane extraction kit, tissue homogenization by Potter-Elvehjem homogenizer in combination with sucrose-cushion ultracentrifugation, and PM enrichment with Tween 40. Moreover, five different protein digestion protocols were applied on the same PM fraction. PM isolation and digestion protocols were evaluated by measuring the amount of transporter proteins by liquid chromatography-tandem mass spectrometry in selected reaction monitoring mode. ResultsMouse liver homogenization by Potter-Elvehjem homogenizer in combination with sucrose-cushion ultracentrifugation and PM enrichment with Tween 40 resulted in two times higher transporter protein quantity (Breast cancer resistance protein (Bcrp) 18.0 fmol/μg protein) in comparison with the PM samples isolated by extraction kit (Bcrp 9.8 fmol/μg protein). The evaluation of protein digestion protocols revealed that the most optimal protocol for PM protein digestion is with Lys-C and trypsin, in combination with trypsin enhancer and heat denaturation. Overall, quantities of Bcrp and Na+/K + ATPase proteins evaluated in mouse liver and kidney cortex by using our optimized PM isolation method, as well as, established digestion protocol were two to three times higher than previously reported and coefficient of variation (CV) for technical replicates was below 10%. ConclusionWe have established an improved transporter protein quantification methodology by optimizing PM isolation and protein digestion procedures. The optimized procedure resulted in a higher transporter protein yield and improved precision.

Comparative Study of References and Protein Quantifications Using Biuret-Spectrophotometric Method

Comparison between the references and the quantification of biuret standard curves can be performed using statistical methods. The objective of this study was to select a method of protein quantification of the biuret-spectrophotometric based on the similar standard curve of the compound. The study used experimental methods in the laboratory and references from a comparative scheme of mean that has categories of statistics called academic and practical terms. Two standard curves were tested with the reference data. The academic way was performed with a comparison of 11.779-12.401% confidence, and the quantification of the results of the protein mean of 8.211% and 10.17% showed no significant difference. The practical methods were carried out with the original reference data of 10.8-12.8%, and the result of the test confidence values of 9.163-11.180% and 7.596-8.826% showed different accuracy results. The quantifications of the protein biuret method displayed different results on how to compare according to academic and practical ways. The quantification method using the Biuret-spectrophotometric practical way shows accuracy by a certain standard curve compared with the original values of references.

Coomassie does it (better): A Robin Hood approach to total protein quantification

Quantitative comparative proteomics require accurate and reproducible assessments of total protein concentration. We report a straightforward, cost-effective adaptation of an established commercial method for total protein quantification (EZQ™), utilising non-proprietary materials and colloidal Coomassie Brilliant Blue (cCBB) staining to achieve greater reproducibility, equal sensitivity, and optimal linearity of signal within a practical concentration range for proteins in common solubilisation buffers (i.e. for isoelectric focussing and/or SDS-PAGE). This method provided more accurate and precise determinations of total protein concentration in human serum prepared for two-dimensional gel electrophoresis, indicating it may be better suited as the lead-in to most quantitative proteomic analyses.

Colorimetric protein determination in microalgae (Chlorophyta): association of milling and SDS treatment for total protein extraction.

The use of colorimetric methods for protein quantification in microalgae is hindered by their elevated amounts of membrane-embedded intracellular proteins. In this work, the protein content of three species of microalgae was determined by the Lowry method after the cells were dried, ball-milled, and treated with the detergent sodium dodecyl sulfate (SDS). Results demonstrated that the association of milling and SDS treatment resulted in a 3- to 7-fold increase in protein quantification. Milling promoted microalgal disaggregation and cell wall disruption enabling access of the SDS detergent to the microalgal intracellular membrane proteins and their efficient solubilization and quantification.

Exosome isolation from distinct biofluids using precipitation and column-based approaches.

The potential of exosomes as biomarker resources for diagnostics, prognostics and even for therapeutics is an area of intense research. Despite the various approaches available, there is no consensus with respect to the best methodology for isolating exosomes and to provide substantial yields with reliable quality. Differential centrifugation is the most commonly used method but it is time-consuming and requires large sample volumes, thus alternative methods are urgently needed. In this study two precipitation-based methods and one column-based approach were compared for exosome isolation from distinct biofluids (serum, plasma and cerebrospinal fluid). Exosome characterization included morphological analyses, determination of particle concentration, stability and exosome preparations’ purity, using different complementary approaches such as Nanoparticle Tracking Analysis, Electrophoretic Light Scattering, Transmission Electron Microscopy, EXOCET colorimetric assay, protein quantification methods and western blotting. The three commercial kits tested successfully isolated exosomes from the biofluids under study, although ExoS showed the best performance in terms of exosome yield and purity. Data shows that methods other than differential centrifugation can be applied to quickly and efficiently isolate exosomes from reduced biofluid volumes. The possibility to use small volumes is fundamental in the context of translational and clinical research, thus the results here presented contribute significantly in this respect.

Using Terminal Transferase-mediated dUTP Nick End-labelling (TUNEL) and Caspase 3/7 Assays to Measure Epidermal Cell Death in Frogs with Chytridiomycosis.

Amphibians are experiencing a great loss in biodiversity globally and one of the major causes is the infectious disease chytridiomycosis. This disease is caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd), which infects and disrupts frog epidermis; however, pathological changes have not been explicitly characterized. Apoptosis (programmed cell death) can be used by pathogens to damage host tissue, but can also be a host mechanism of disease resistance for pathogen removal. In this study, we quantify epidermal cell death of infected and uninfected animals using two different assays: terminal transferase-mediated dUTP nick end-labelling (TUNEL), and caspase 3/7. Using ventral, dorsal, and thigh skin tissue in the TUNEL assay, we observe cell death in the epidermal cells in situ of clinically infected animals and compare cell death with uninfected animals using fluorescent microscopy. In order to determine how apoptosis levels in the epidermis change over the course of infection we remove toe-tip samples fortnightly over an 8-week period, and use a caspase 3/7 assay with extracted proteins to quantify activity within the samples. We then correlate caspase 3/7 activity with infection load. The TUNEL assay is useful for localization of cell death in situ, but is expensive and time intensive per sample. The caspase 3/7 assay is efficient for large sample sizes and time course experiments. However, because frog toe tip biopsies are small there is limited extract available for sample standardization via protein quantification methods, such as the Bradford assay. Therefore, we suggest estimating skin surface area through photographic analysis of toe biopsies to avoid consuming extracts during sample standardization.

A novel nano-immunoassay method for quantification of proteins from CD138-purified myeloma cells: biological and clinical utility

Protein analysis in bone marrow samples from patients with multiple myeloma has been limited by the low concentration of proteins obtained after CD138+ cell selection. A novel approach based on capillary nano-immunoassay could make it possible to quantify dozens of proteins from each myeloma sample in an automated manner. Here we present a method for the accurate and robust quantification of the expression of multiple proteins extracted from CD138-purified multiple myeloma samples frozen in RLT Plus buffer, which is commonly used for nucleic acid preservation and isolation. Additionally, the biological and clinical value of this analysis for a panel of 12 proteins essential to the pathogenesis of multiple myeloma was evaluated in 63 patients with newly diagnosed multiple myeloma. The analysis of the prognostic impact of CRBN/Cereblon and IKZF1/Ikaros mRNA/protein showed that only the protein levels were able to predict progression-free survival of patients; mRNA levels were not associated with prognosis. Interestingly, high levels of Cereblon and Ikaros proteins were associated with longer progression-free survival only in patients who received immunomodulatory drugs and not in those treated with other drugs. In conclusion, the capillary nano-immunoassay platform provides a novel opportunity for automated quantification of the expression of more than 20 proteins in CD138+ primary multiple myeloma samples.

Reducing the risk of iatrogenic Creutzfeldt–Jakob disease by improving the cleaning of neurosurgical instruments

In all, there have been 178 variant Creutzfeldt-Jakob disease (vCJD) patients diagnosed in the UK, with an estimated maximum 1:2000 carriage rate based on archived appendix and tonsil tissue, implying that infection may be rare but carriage relatively frequent. Previous workers have identified that maintenance of surgical instruments in a humid atmosphere after use and prior to cleaning assists cleaning efficacy. Recently the Department of Health/Advisory Committee on Dangerous Pathogens UK have recommended a surgical instrument cleanliness threshold post cleaning of <5μg protein per instrument side. To quantify cleanliness of neurosurgical instruments and to investigate cost-effective measures for improved cleaning. Two instrument protein quantification methods were used: one based on the International Standard (15883 series) using sodium dodecyl sulphate elution and ortho-phthalaldehyde reaction, and a second in-situ protein fluorescence detection system (ProReveal) providing results per instrument side. In-vitro investigation of the efficacy of some commercial and in-house pre-clean wetting agents was undertaken using artificial test soil and stainless steel discs under standard conditions. In-vivo evaluation of best-performing in-vitro agents was undertaken on craniotomy sets. ProReveal technology demonstrated that 163 out of 187 (87%) neurosurgical instruments had <5μg residual protein per instrument side. The use of proprietary National Health Service plastic bags and sterile water-soaked wound pads were equivalent in efficacy to commercial pre-cleaning wetting products and significantly less expensive. Although we demonstrate low in-situ protein levels on neurosurgical instruments and the beneficial effects of keeping instruments moist, other cleaning critical-control points such as instrument loading patterns should also be monitored.

Biuret and Bradford Methods Suitability for Protein Quantification in Rapeseed Meal

Summary Being attractive for insects, non-genetically modified rapeseed is valuable for maintaining environmental biodiversity. Primarily, the rapeseed is an important industrial crop which is used for production of vegetable oil. Oil extraction from rapeseeds results in the generation of substantial amounts of rapeseed meal which is used either as a protein rich feed additive or as a source for preparation of protein containing ingredients for food industry. Both applications require frequent evaluation of protein content. Although Kjeldahl method is considered standard, it is not appropriate for routine evaluation of protein content in protein extracts. The aim of the study was to evaluate suitability of biuret and Bradford methods for protein quantification in rapeseed meal extracts. After consecutive triple extraction of proteins with water, 5% NaCl, 70% ethyl alcohol and 0.1 N NaOH, protein evaluation of each albumin, globulin, prolamin and glutelin extraction aliquot demonstrated overall lower protein content by Bradford method compared to biuret method. The most pronounced differences in protein content were observed with prolamin fraction where three fold higher protein concentrations in each extraction aliquot was observed when biuret method was applied for the evaluation. Comparative quantification of the total protein of each of the four fractions followed a similar trend of lower protein content evaluation by Bradford method. Overall results indicated biuret method as more suitable for protein quantification in rapeseed meal extracts which was confirmed by comparison with data obtained by Kjeldahl method.

A Proximity Extension Assay (PEA)-based method for quantification of bevacizumab

IntroductionProximity Extension Assay (PEA) is a direct one-step protein quantification method using a pair of DNA oligonucleotides linked to antibodies against the target molecule. It requires polyclonal or two monoclonal antibodies (mAbs) that bind to target epitopes close enough to form a DNA duplex which is quantified by real-time PCR. Bevacizumab, an anti-cancer drug, is a mAb against vascular endothelial growth factor with common cardiovascular adverse effects. It is widely used off-label to treat neovascular eye disorders by intravitreal application of small doses. Even then, certain amount reaches systemic circulation which is considered relevant regarding safety. We aimed to set-up a PEA-based assay for bevacizumab in human plasma and to preliminary evaluate it in patients treated intravitreally. MethodsWe tested (PEA, quantitative PCR) several combinations of commercial mAbs and a Fab fragment against bevacizumab. The best combination was used to quantify bevacizumab in three patients donating plasma before and 24 h after the first intravitreal injection. ResultsA combination of a mAb and a Fab fragment (HCA184 and HCA182, Bio-Rad Laboratories, Inc.) performed best: standard curve R2 0.98, linear dynamic range 1–1000 pM, lower limit of quantification 1 pM (149 pg/mL) and a satisfactory precision (coefficient of variation 12%). All pre-dose patient concentrations were zero, while post-dose concentrations were 10.94, 13.73 and 55.49 ng/mL, in line with previous reports. DiscussionThis is the first set-up of a PEA-based assay for quantification of bevacizumab in human plasma. Its good performance and high sensitivity support further evaluation for potential uses particularly when the expected concentrations are low.

Multi-laboratory assessment of reproducibility, qualitative and quantitative performance of SWATH-mass spectrometry

Quantitative proteomics employing mass spectrometry is an indispensable tool in life science research. Targeted proteomics has emerged as a powerful approach for reproducible quantification but is limited in the number of proteins quantified. SWATH-mass spectrometry consists of data-independent acquisition and a targeted data analysis strategy that aims to maintain the favorable quantitative characteristics (accuracy, sensitivity, and selectivity) of targeted proteomics at large scale. While previous SWATH-mass spectrometry studies have shown high intra-lab reproducibility, this has not been evaluated between labs. In this multi-laboratory evaluation study including 11 sites worldwide, we demonstrate that using SWATH-mass spectrometry data acquisition we can consistently detect and reproducibly quantify >4000 proteins from HEK293 cells. Using synthetic peptide dilution series, we show that the sensitivity, dynamic range and reproducibility established with SWATH-mass spectrometry are uniformly achieved. This study demonstrates that the acquisition of reproducible quantitative proteomics data by multiple labs is achievable, and broadly serves to increase confidence in SWATH-mass spectrometry data acquisition as a reproducible method for large-scale protein quantification.

Prediction of the genetic similarity of wheat and wheat quality by reversed-phase high-performance liquid chromatography and lab-on-chip methods

The aim of this study was to compare efficiency of RP-HPLC (Reversed-Phase High-Performance Liquid Chromatography) and LOC (Lab-on-Chip) methods for wheat gluten protein quantification regarding clustering of wheat cultivars according to the genetic similarity (HMW-GS combinations), as well as to explore relations of these two methods to wheat quality parameters. For that purpose, wheat quality parameters (protein content, falling number, wet gluten content, gluten index, Farinograph, Extensograph, and Amylograph), as well as amounts of gliadin and glutenin fractions by RP-HPLC and LOC methods were determined in two different sets of wheat cultivars (Croatian and Serbian). The percentages of gluten proteins and the values of quality parameters were used to characterize the samples by principal component analysis (PCA). Gluten protein quantification performed by method based on the protein fraction separation by molecular weights (LOC) was better for grouping of genetically similar wheat cultivars than quantification of proteins separated by their different solubility in specified solvent gradient (RP-HPLC). LOC method showed higher potential in wheat quality prediction.

Quantification of dystrophin to assess the effects of dystrophin-restoring treatments in Duchenne muscular dystrophy: Lessons from the development of eteplirsen

Objective: Accurate assessment of dystrophin expression is essential in evaluating effects of dystrophin-restoring therapies. Eteplirsen was recently approved for patients with Duchenne muscular dystrophy (DMD) amenable to exon 51 skipping based on an observed increase in dystrophin. This analysis used 3 complementary methods for dystrophin protein quantification in muscle tissue at baseline and following eteplirsen treatment at Week 180 of a clinical trial (NCT01540409). Methods: Three well-controlled methods were used to assess changes from baseline in dystrophin: Western blot analysis to quantify total dystrophin protein (Sarepta), pathologist scoring of immunofluorescent staining to determine percent dystrophin-positive fibers (PDPF; Flagship Biosciences, NCH), and immunofluorescence to assess dystrophin-associated fiber intensity (NCH). All analyses were performed using blinded conditions. The validated Western blot method was quantitative and optimized to a low linear dynamic range expected for DMD, with no saturating signal. Reproducible evaluation of PDPF analysis, as evidenced by high interrater reliability, was performed independently by 3 trained pathologists (Flagship Biosciences) using standardized viewing conditions and scoring requirements. Pathologists effectively demonstrated and maintained concordance throughout scoring. Analysis of immunofluorescent dystrophin fiber intensity was performed on 4 systematic, random subsample images of each tissue section using BIOQUANT™ software. Results: In total, 20 biopsies were evaluated (eteplirsen patients, n=11; untreated DMD controls, n=9). At Week 180, compared with untreated control biopsies, significantly greater dystrophin expression was observed in eteplirsen-treated patient samples by Western blot (absolute difference, +0.85%; 11.6-fold greater; P=.007), PDPF manual scoring (absolute difference, +16.3%; 15.5-fold greater; P<.001), and dystrophin immunofluorescent fiber intensity (absolute difference, +13.2%; 2.4-fold greater; P<.001). Conclusion: Our findings establish the importance of using a combination of carefully optimized, complementary methods for dystrophin quantification in muscle biopsies of DMD patients. Refinements of the methods are discussed, with the goal of improving sensitivity and reducing potential bias.

PH Readout enhanced ELISA for point-of-care testing of cardiac troponin I

Enzyme-linked immunosorbent assay (ELISA) as a conventional method for protein quantification has its characteristic properties, however, it is challenging to implement excellent portability and sensitivity at the same time. In this study, we described a pH ELISA using synthetic melanin nanoparticles (SMNPs) for the co-immobilization of glucose oxidase (GOx) and second antibody (Ab2) as signal labels, portable pH meter as signal readout device for point-of-care testing (POCT) of cardiac troponin I (cTnI). In accordance with the varying amount of cTnI, following sandwich type immunoassay, proportional SMNPs-GOx-Ab2 were immobilized specifically resulting in corresponding decrease of pH values owing to GOx loaded on SMNPs can high-efficiency convert glucose into gluconic acid. This assay is easy-to-use, portable, sensitive and able to realize POCT, affording a linear range from 0.5pg/mL to 10ng/mL and low limit of detection (LOD) of 0.15pg/mL towards cTnI, which was demonstrated the significant promising in the early diagnosis and screening of acute myocardial infarction.

Development of a validated Western blot method for quantification of human dystrophin protein used in Phase II and III clinical trials of eteplirsen for the treatment of Duchenne muscular dystrophy (DMD) (P5.105)

Development of a validated Western blot method for quantification of human dystrophin protein used in Phase II and III clinical trials of eteplirsen for the treatment of Duchenne muscular dystrophy (DMD) (P5.105)

Experimental design and data-analysis in label-free quantitative LC/MS proteomics: A tutorial with MSqRob

Label-free shotgun proteomics is routinely used to assess proteomes. However, extracting relevant information from the massive amounts of generated data remains difficult. This tutorial provides a strong foundation on analysis of quantitative proteomics data. We provide key statistical concepts that help researchers to design proteomics experiments and we showcase how to analyze quantitative proteomics data using our recent free and open-source R package MSqRob, which was developed to implement the peptide-level robust ridge regression method for relative protein quantification described by Goeminne et al. MSqRob can handle virtually any experimental proteomics design and outputs proteins ordered by statistical significance. Moreover, its graphical user interface and interactive diagnostic plots provide easy inspection and also detection of anomalies in the data and flaws in the data analysis, allowing deeper assessment of the validity of results and a critical review of the experimental design. Our tutorial discusses interactive preprocessing, data analysis and visualization of label-free MS-based quantitative proteomics experiments with simple and more complex designs. We provide well-documented scripts to run analyses in bash mode on GitHub, enabling the integration of MSqRob in automated pipelines on cluster environments (https://github.com/statOmics/MSqRob). SignificanceThe concepts outlined in this tutorial aid in designing better experiments and analyzing the resulting data more appropriately. The two case studies using the MSqRob graphical user interface will contribute to a wider adaptation of advanced peptide-based models, resulting in higher quality data analysis workflows and more reproducible results in the proteomics community. We also provide well-documented scripts for experienced users that aim at automating MSqRob on cluster environments.

A curated gluten protein sequence database to support development of proteomics methods for determination of gluten in gluten-free foods

The unique physiochemical properties of wheat gluten enable a diverse range of food products to be manufactured. However, gluten triggers coeliac disease, a condition which is treated using a gluten-free diet. Analytical methods are required to confirm if foods are gluten-free, but current immunoassay-based methods can unreliable and proteomic methods offer an alternative but require comprehensive and well annotated sequence databases which are lacking for gluten. A manually a curated database (GluPro V1.0) of gluten proteins, comprising 630 discrete unique full length protein sequences has been compiled. It is representative of the different types of gliadin and glutenin components found in gluten. An in silico comparison of their coeliac toxicity was undertaken by analysing the distribution of coeliac toxic motifs. This demonstrated that whilst the α-gliadin proteins contained more toxic motifs, these were distributed across all gluten protein sub-types. Comparison of annotations observed using a discovery proteomics dataset acquired using ion mobility MS/MS showed that more reliable identifications were obtained using the GluPro V1.0 database compared to the complete reviewed Viridiplantae database. This highlights the value of a curated sequence database specifically designed to support the proteomic workflows and the development of methods to detect and quantify gluten. SignificanceWe have constructed the first manually curated open-source wheat gluten protein sequence database (GluPro V1.0) in a FASTA format to support the application of proteomic methods for gluten protein detection and quantification. We have also analysed the manually verified sequences to give the first comprehensive overview of the distribution of sequences able to elicit a reaction in coeliac disease, the prevalent form of gluten intolerance. Provision of this database will improve the reliability of gluten protein identification by proteomic analysis, and aid the development of targeted mass spectrometry methods in line with Codex Alimentarius Commission requirements for foods designed to meet the needs of gluten intolerant individuals.