Recent developments in Phos-tag electrophoresis for the analysis of phosphoproteins in proteomics.

INTRODUCTION: Phosphate-binding tag (Phos-tag) sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is an important development capable of analyzing the phosphorylation state of proteins. Conventionally, proteins were separated via SDS-PAGE and Phos-tag SDS-PAGE that use different gels to identify phosphorylated proteins. However, it was often difficult to compare the electrophoretic mobility of the proteins in the different gels used. The recently developed Phos-tag diagonal electrophoresis has been able to solve this problem. It can indicate the SDS-PAGE and Phos-tag SDS-PAGE patterns on a single gel; therefore, phosphorylated proteins can be distinguished easily from non-phosphorylated proteins. AREAS COVERED: This review assesses the importance of Phos-tag electrophoresis, which enables the analysis of protein phosphorylation states, in the field of proteomics. Additionally, this review describes the significance and actual experimental technique of Phos-tag diagonal electrophoresis, which was recently developed to overcome the drawbacks of Phos-tag SDS-PAGE. EXPERT OPINION: Although shotgun analysis of proteins allows detecting many phosphorylation sites, it is challenging to clarify the differences in the phosphorylation states of protein molecules using this technique. Therefore, Phos-tag SDS-PAGE is frequently used to determine the phosphorylation state of proteins. This technique has become more powerful with the recent development of Phos-tag diagonal electrophoresis.Abbreviations: BIS, N,N'-methylenebis(acrylamide); CBB, Coomassie brilliant blue R250; ESI, electrospray ionization; hnRNP, heterogeneous ribonucleoprotein K; LTQ-Orbitrap, Linear trap quadrupole-Orbitrap; LC, liquid chromatography; MS, mass spectrometry; MALDI, matrix-assisted laser desorption ionization; Phos-tag, phosphate-binding tag [1,3-bis [bis (pyridine-2-ylmethyl) amino] propane-2-olate]; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TOF, time of flight; 2D-DIGE, fluorescence-labeled two-dimensional difference gel electrophoresis; 2-DE, two-dimensional gel electrophoresis.

An optimized SDS-PAGE protocol with a new blotting system for the initial testing procedure of ESAs in doping control.

Recombinant erythropoietins (rEPOs) are still among the substances endurance athletes use for doping. Detection methods are based on an electrophoretic separation of the proteins followed by a western blot and immunodetection with specific anti-EPO antibodies. In addition to IEF-PAGE, the SDS-PAGE method has been used to differentiate endogenous EPO from rEPOs by their molecular weight (MW). However, to adapt to new generations of rEPOs exhibiting higher MW, which were not well detected after SDS-PAGE, sodium lauroyl sarcosinate (SAR) is now used instead of sodium dodecyl sulfate (SDS) for the initial EPO testing procedure on doping control samples. The SAR-PAGE method is nevertheless expensive as it requires frequent buffer preparations using highly purified sarkosyl powder. In addition, this reagent needs to be handled with care due to acute toxicity by inhalation. The aim of this work was to improve the SDS-PAGE method by increasing its sensitivity and transfer of high-MW rEPOs. First, using a biotinylated primary anti-EPO antibody and avoiding the use of a secondary antibody increased the general sensitivity of both SDS-PAGE and SAR-PAGE to all rEPOs about four-fold. Then, by changing the buffer system during the protein transfer, with a CAPS buffer and a discontinuous buffer transfer system, high-MW rEPOs, EPO-Fc and CERA were transferred with higher efficiency and detected with high sensitivity. This optimized SDS-PAGE protocol could be adopted by anti-doping laboratories as an alternative to SAR-PAGE.

Detection of recombinant erythropoietin biosimilar Jimaixin™ after administration in healthy subjects.

Jimaixin™ (Jintan Ltd, China) is a biosimilar of recombinant erythropoietin (rEPO) now authorized for therapeutic application in China. With a risk of abuse by athletes, a clear evaluation of its detection using the electrophoretic methods in use in antidoping laboratories was necessary. In a previous work, we showed that Jimaixin™ electrophoretic profile presented slight changes compared with the original drug (first generation rEPO) and that a spike of Jimaixin™ in urine and serum was well identified by SDS-PAGE but with less performance by IEF-PAGE unless a neuraminidase treatment was applied first. The aims of this research were to perform an intravenous administration of Jimaixin™ on three healthy subjects (one microdose [10 IU/kg] and three therapeutic doses [50 IU/kg]) and to evaluate the detection in urine and blood up to 7 days post administration. Analysis of the samples showed that Jimaixin™ detection was complicated by IEF-PAGE due to the loss of the most distinctive basic isoforms. In addition, a neuraminidase treatment did not improve detection (contrary to the observations from spike experiments). On the contrary, Jimaixin™ was very efficiently detected in blood and urine by SDS-PAGE: up to 40 h after a microdose and up to 7 days after the therapeutic doses. The effect of Jimaixin™ on hematological parameters was limited to a clear but transitory increase of the reticulocytes. These data give new elements to better survey a potential misuse of Jimaixin™ by athletes.

Production and Partial Characterization of α-Amylase Enzyme from Marine Actinomycetes.

Amylase producing actinobacteria were isolated and characterized from terrestrial environment. There are a limited number of reports investigating the marine environment; hence, in the present study, four marine enzymes were tested for their amylase production ability. On starch agar plates, the Streptomyces rochei strain showed a higher hydrolytic zone (24 mm) than the other isolates. Growth under optimized culture conditions using Plackett-Burman's experimental design led to a 1.7, 9.8, 7.7, and 3.12-fold increase for the isolates S. griseorubens, S. rochei, S. parvus, and Streptomyces sp., respectively, in the specific activity measurement. When applying the Box-Behnken design on S. rochei using the most significant parameters (starch, K2HPO4, pH, and temperature), there was a 12.22-fold increase in the specific activity measurement 7.37 U/mg. The α-amylase was partially purified, and its molecular weight was determined using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. α-Amylase was particularly active at pH 6 and 65°C. The purified enzyme was most active at 65°C and pH 6, thermal stability of 70°C for 40 min, and salt concentration of 1 M with Km and Vmax of 6.58 mg/ml and 21.93 µmol/ml/min, respectively. The α-amylase was improved by adding Cu+2, Zn+2, and Fe+2 (152.21%, 207.24%, and 111.89%). Increased production of α-amylase enzyme by S. rochei KR108310 leads to production of significant industrial products.

A Fast and Reliable Process to Fabricate Regenerated Silk Fibroin Solution from Degummed Silk in 4 Hours.

Silk fibroin has a high potential for use in several approaches for technological and biomedical applications. However, industrial production has been difficult to date due to the lengthy manufacturing process. Thus, this work investigates a novel procedure for the isolation of non-degraded regenerated silk fibroin that significantly reduces the processing time from 52 h for the standard methods to only 4 h. The replacement of the standard degumming protocol by repeated short-term microwave treatments enabled the generation of non-degraded degummed silk fibroin. Subsequently, a ZnCl2 solution was used to completely solubilize the degummed fibroin at only 45 °C with an incubation time of only 1 h. Desalting was performed by gel filtration. Based on these modifications, it was possible to generate a cytocompatible aqueous silk fibroin solution from degummed silk within only 4 h, thus shortening the total process time by 48 h without degrading the quality of the isolated silk fibroin solution.

A Rapid, Reliable RP-UPLC Method for Large-Scale Analysis of Wheat HMW-GS Alleles.

High-molecular-weight glutenin subunits (HMW-GS) account for only 10% of total wheat storage proteins, but play an important role in the processing quality of wheat flour. Therefore, identifying HMW-GS alleles associated with good end-use quality provides important information for wheat breeders. To rapidly, accurately and reproducibly identify HMW-GS, we established an optimized reversed-phase ultra-performance liquid chromatography (RP-UPLC) method. Separation parameters were optimized using an ACQUITY UPLC Protein BEH C4 column and stepwise ACN gradient, and the separation patterns and retention times (RTs) of 22 subunits were comparatively analyzed in 16 standard wheat cultivars. All HMW-GS proteins were well separated within about 5.5 min, and all analyses were complete within 12 min. We distinguished the 16 subunits based on RT, although three subunits in 1Bx (1Bx7/1Bx7OE and 1Bx17) and three subunits in 1By (1By8*, 1By9 and 1By15) had overlapping RTs; these were differentiated by SDS-PAGE. To distinguish 1Bx7 and 1Bx7OE, which differ in protein abundance, RP-UPLC was combined with PCR analysis of DNA junction markers. The optimized method was successfully applied to determine HMW-GS alleles in a large collection of bread wheat germplasm (1787 lines). This protocol is an appropriate option for selecting lines harboring favorable HMW-GS alleles in wheat breeding.

Distinct phosphorylation profiles of tau in brains of patients with different tauopathies.

Tauopathies are neurodegenerative diseases that are characterized by pathological accumulation of tau protein. Tau is hyperphosphorylated in the brain of tauopathy patients, and this phosphorylation is proposed to play a role in disease development. However, it has been unclear whether phosphorylation is different among different tauopathies. Here, we investigated the phosphorylation states of tau in several tauopathies, including corticobasal degeneration, Pick's disease, progressive supranuclear palsy (PSP), argyrophilic grain dementia (AGD) and Alzheimer's disease (AD). Analysis of tau phosphorylation profiles using Phos-tag SDS-PAGE revealed distinct phosphorylation of tau in different tauopathies, whereas similar phosphorylation patterns were found within the same tauopathy. For PSP, we found 2 distinct phosphorylation patterns suggesting that PSP may consist of 2 different related diseases. Immunoblotting with anti-phospho-specific antibodies showed different site-specific phosphorylation in the temporal lobes of patients with different tauopathies. AD brains showed increased phosphorylation at Ser202, Thr231 and Ser235, Pick's disease brains showed increased phospho-Ser202, and AGD brains showed increased phospho-Ser396. The cis conformation of the peptide bond between phospho-Thr231 and Pro232 (cis ptau) was increased in AD and AGD. These results indicate that while tau is differently phosphorylated in tauopathies, a similar pathological mechanism may occur in AGD and AD patients. The present data provide useful information regarding tau pathology and diagnosis of tauopathies.

Isolation of HDL by sequential flotation ultracentrifugation followed by size exclusion chromatography reveals size-based enrichment of HDL-associated proteins.

High-density lipoprotein (HDL) particles have multiple beneficial and cardioprotective roles, yet our understanding of their full structural and functional repertoire is limited due to challenges in separating HDL particles from contaminating plasma proteins and other lipid-carrying particles that overlap HDL in size and/or density. Here we describe a method for isolating HDL particles using a combination of sequential flotation density ultracentrifugation and fast protein liquid chromatography with a size exclusion column. Purity was visualized by polyacrylamide gel electrophoresis and verified by proteomics, while size and structural integrity were confirmed by transmission electron microscopy. This HDL isolation method can be used to isolate a high yield of purified HDL from a low starting plasma volume for functional analyses. This method also enables investigators to select their specific HDL fraction of interest: from the least inclusive but highest purity HDL fraction eluting in the middle of the HDL peak, to pooling all of the fractions to capture the breadth of HDL particles in the original plasma sample. We show that certain proteins such as lecithin cholesterol acyltransferase (LCAT), phospholipid transfer protein (PLTP), and clusterin (CLUS) are enriched in large HDL particles whereas proteins such as alpha-2HS-glycoprotein (A2HSG), alpha-1 antitrypsin (A1AT), and vitamin D binding protein (VDBP) are enriched or found exclusively in small HDL particles.

Characterization of the Free and Membrane-Associated Fractions of the Thylakoid Lumen Proteome in Arabidopsis thaliana.

The thylakoid lumen houses proteins that are vital for photosynthetic electron transport, including water-splitting at photosystem (PS) II and shuttling of electrons from cytochrome b6f to PSI. Other lumen proteins maintain photosynthetic activity through biogenesis and turnover of PSII complexes. Although all lumen proteins are soluble, these known details have highlighted interactions of some lumen proteins with thylakoid membranes or thylakoid-intrinsic proteins. Meanwhile, the functional details of most lumen proteins, as well as their distribution between the soluble and membrane-associated lumen fractions, remain unknown. The current study isolated the soluble free lumen (FL) and membrane-associated lumen (MAL) fractions from Arabidopsis thaliana, and used gel- and mass spectrometry-based proteomics methods to analyze the contents of each proteome. These results identified 60 lumenal proteins, and clearly distinguished the difference between the FL and MAL proteomes. The most abundant proteins in the FL fraction were involved in PSII assembly and repair, while the MAL proteome was enriched in proteins that support the oxygen-evolving complex (OEC). Novel proteins, including a new PsbP domain-containing isoform, as well as several novel post-translational modifications and N-termini, are reported, and bi-dimensional separation of the lumen proteome identified several protein oligomers in the thylakoid lumen.

Utilizing SARS-CoV-2 to teach PCR and gel electrophoresis in a pair of asynchronous distant learning laboratory exercises.

Amidst the COVID-19 pandemic, many colleges shifted to online learning, creating a need for teaching materials that could be deployed in the online setting. A pair of virtual laboratory exercises with a COVID-19 theme were created for first year Biology majors to introduce students to the topics of polymerase chain reaction and gel electrophoresis. The exercises were effective in promoting student learning of both topics in an online asynchronous setting, and could easily be adapted for use in other courses or in a synchronous online setting.

Analysis of Murein Hydrolases and Proteases Through Zymography.

Zymography has been used to analyze enzymatic activity and processing of enzymes for many years. We have used bacterial cells copolymerized into the acrylamide gel to analyze specific activity of murein hydrolases of interest. In addition, this method has been widely used to examine and distinguish protease activities using different substrates. This chapter provides instruction for zymography of both extracellular murein hydrolases and proteases produced by Staphylococcus aureus.

Unveiling Putative Functions of Mucus Proteins and Their Tryptic Peptides in Seven Gastropod Species Using Comparative Proteomics and Machine Learning-Based Bioinformatics Predictions.

Gastropods are among the most diverse animals. Gastropod mucus contains several glycoproteins and peptides that vary by species and habitat. Some bioactive peptides from gastropod mucus were identified only in a few species. Therefore, using biochemical, mass spectrometric, and bioinformatics approaches, this study aimed to comprehensively identify putative bioactive peptides from the mucus proteomes of seven commonly found or commercially valuable gastropods. The mucus was collected in triplicate samples, and the proteins were separated by 1D-SDS-PAGE before tryptic digestion and peptide identification by nano LC-MS/MS. The mucus peptides were subsequently compared with R scripts. A total of 2818 different peptides constituting 1634 proteins from the mucus samples were identified, and 1218 of these peptides (43%) were core peptides found in the mucus of all examined species. Clustering and correspondence analyses of 1600 variable peptides showed unique mucous peptide patterns for each species. The high-throughput k-nearest neighbor and random forest-based prediction programs were developed with more than 95% averaged accuracy and could identify 11 functional categories of putative bioactive peptides and 268 peptides (9.5%) with at least five to seven bioactive properties. Antihypertensive, drug-delivering, and antiparasitic peptides were predominant. These peptides provide an understanding of gastropod mucus, and the putative bioactive peptides are expected to be experimentally validated for further medical, pharmaceutical, and cosmetic applications.

Soluble Prokaryotic Overexpression and Purification of Human GM-CSF Using the Protein Disulfide Isomerase b'a' Domain.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a member of the colony-stimulating factor (CSF) family, which functions to enhance the proliferation and differentiation of hematopoietic stem cells and other hematopoietic lineages such as neutrophils, dendritic cells, or macrophages. These proteins have thus generated considerable interest in clinical therapy research. A current obstacle to the prokaryotic production of human GM-CSF (hGM-CSF) is its low solubility when overexpressed and subsequent complex refolding processes. In our present study, the solubility of hGM-CSF was examined when combined with three N-terminal fusion tags in five E. coli strains at three different expression temperatures. In the five E. coli strains BL21 (DE3), ClearColi BL21 (DE3), LOBSTR, SHuffle T7 and Origami2 (DE3), the hexahistidine-tagged hGM-CSF showed the best expression but was insoluble in all cases at each examined temperature. Tagging with the maltose-binding protein (MBP) and the b'a' domain of protein disulfide isomerase (PDIb'a') greatly improved the soluble overexpression of hGM-CSF at 30 °C and 18 °C. The solubility was not improved using the Origami2 (DE3) and SHuffle T7 strains that have been engineered for disulfide bond formation. Two conventional chromatographic steps were used to purify hGM-CSF from the overexpressed PDIb'a'-hGM-CSF produced in ClearColi BL21 (DE3). In the experiment, 0.65 mg of hGM-CSF was isolated from a 0.5 L flask culture of these E. coli and showed a 98% purity by SDS-PAGE analysis and silver staining. The bioactivity of this purified hGM-CSF was measured at an EC50 of 16.4 ± 2 pM by a CCK8 assay in TF-1 human erythroleukemia cells.

Purification and characterization of angiotensin-converting enzyme (ACE) from sheep lung.

Angiotensin-converting enzyme (ACE, EC 3.4.15.1) in the renin-angiotensin system regulates blood pressure by catalyzing angiotensin I to the vasoconstrictor angiotensin II. In this study, the ACE was purified and characterized from sheep lung. The kinetic properties of the ACE were designated. The inhibition effect of captopril, a specific ACE inhibitor, was determined. ACE was purified from sheep lung using the affinity chromatography method in one step. NHS-activated Sepharose 4 Fast Flow as column filler and lisinopril as a ligand in this method used. The molecular weight and purity of ACE were designated using the SDS-PAGE method. Optimum temperature and optimum pH were found for purified ACE. KM and Vmax values from Lineweaver-Burk charts determined. The inhibition type, IC50, and Ki values of captopril on purified ACE were identified. ACE was 6405-fold purified from sheep lung by affinity chromatography in one step and specific activity was 16871 EU/mg protein. The purity and molecular weight of ACE were found with SDS-PAGE and observed two bands at around 60 kDa and 70 kDa on the gel. Optimum temperature and optimum pH were designated for purified ACE. Optimum temperature and pH were found as 40 °C and pH 7.4, respectively. Vmax and KM values were calculated to be 35.59 (µmol/min).mL-1 and 0.18 mM, respectively. IC50 value of captopril was found as 0.51 nM. The inhibition type of captopril was determined as non-competitive from the Lineweaver-Burk graph and the Ki value was 0.39 nM. As a result, it was observed in this study that the ACE enzyme can be successfully purified from sheep lungs in one step. Also, it was determined that captopril, which is a specific ACE inhibitor, has a significant inhibitory effect with a very low IC50 value of 0.51 nM.

Coexpression and Copurification of RNA-Protein Complexes in Escherichia coli.

For structural, biochemical, or pharmacological studies, it is required to have pure RNA in large quantities. We previously devised a generic approach that allows for efficient in vivo expression of recombinant RNA in Escherichia coli. We have extended the "tRNA scaffold" method to RNA-protein coexpression in order to express and purify RNA by affinity in native condition. As a proof of concept, we present the expression and the purification of the AtRNA-mala in complex with the MS2 coat protein.

Design and synthesis of a novel nanocomposite based on magnetic dopamine nanoparticles for purification of α-amylase from the bovine milk.

In this paper, a novel nanocomposite based on magnetic nanoparticles decorated by dopamine were reported. Three modified magnetic nanocomposites by dopamine were offered with different type of linkers. The mentioned magnetic nanocomposites were applied to separate α-amylase protein from fresh bovine milk. All of the magnetic nanocomposites were characterized and investigated by using Fourier-transform infrared spectroscopy, energy-dispersive X-ray spectroscopy, field-emission scanning microscope, X-ray diffraction pattern, and vibrating-sample magnetometer analyses. To investigate the purifying application, sodium dodecyl sulfate polyacrylamide gel electrophoresis, one-dimensional isoelectric focusing gel electrophoresis, and alpha-amylase activity assay were employed. With paying attention to factors such as yield of purification and concentration of separated protein by each of magnetic nanocomposite, it could be concluded that the length of linkers played an important role in α-amylase protein separation. According to the results, the best separation and purification of α-amylase protein with 49.83% recovery and 40.11-fold purification efficiency was related to longest length linker, 1,4-butanediol diglycidyl ether, because of considerable conjugation with nanocomposite. Also, docking calculation has shown that the binding energy is - 1.697 kcal/mol and ΔG = - 6.844 kcal/mol which result that the interaction process between dopamine and α-amylase protein is spontaneous.

Native Gel Electrophoresis and Immunoblotting to Analyze Electron Transport Chain Complexes.

Native electrophoresis is a powerful tool to analyze the mitochondrial electron transport chain complexes (Cx) I-V and their assembly into supercomplexes. Valuable information regarding the composition and bioenergetic regulation in physiological and pathological conditions can be obtained. This chapter compares different types of native electrophoresis to analyze mitochondrial supercomplexes.

A rapid method for protein staining in polyacrylamide gels using water saturated with chloroform.

Polyacrylamide gel electrophoresis, followed by an appropriate staining, is a popular and useful analytical procedure for protein identification and characterization. The aim of this study was to develop a method for protein visualization in polyacrylamide gels that would be alternative to Coomassie blue or silver staining. The proposed method is simple, fast and inexpensive. The optimized protocol for protein staining and visualization takes as little as 6 minutes and utilizes deionized water and chloroform. Fluorescence of proteins is induced by UV light and can be detected with a standard transilluminator.

Zymography for Picogram Detection of Lipase and Esterase Activities.

Lipases and esterases are important catalysts with wide varieties of industrial applications. Although many methods have been established for detecting their activities, a simple and sensitive approach for picogram detection of lipolytic enzyme quantity is still highly desirable. Here we report a lipase detection assay which is 1000-fold more sensitive than previously reported methods. Our assay enables the detection of as low as 5 pg and 180 pg of lipolytic activity by direct spotting and zymography, respectively. Furthermore, we demonstrated that the detection sensitivity was adjustable by varying the buffering capacity, which allows for screening of both high and low abundance lipolytic enzymes. Coupled with liquid chromatography-mass spectrometry, our method provides a useful tool for sensitive detection and identification of lipolytic enzymes.

An improved acetic acid-urea polyacrylamide electrophoresis method to evaluate bovine sperm protamines.

The acetic acid-urea polyacrylamide gel electrophoresis system could separate very similar basic proteins on differences in size and effective charge. This system has been used for many years to analyse histones and their post-translational modifications and widely used in the study of mammal protamines. Two types of protamine have been described, the protamine 1 (P1) and the protamine 2 (P2) family members, which are synthetized by PRM1 and PRM2 genes. The ratio of P1 and P2 is important for predicting fertility in humans and mice. Therefore, the quantification of protamines is a fundamental step in order to establish the ratio between P1 and P2 in these species. In other mammals, studies linking sperm protamination and the protamine ratio with fertility are increasing. So, the use of an effective technique to separate and quantify protamines is important to study sperm P1/P2 ratio. Therefore, this article describes in detail a feasible and useful procedure to isolate bovine sperm protamines, to perform pre-electrophoresis with PEG solution and finally to carry out acid-urea polyacrylamide gel electrophoresis in reverse polarity. This technique allows a clear separation and efficient detection of bovine sperm protamines.