Identifying a Ubiquitinated Adaptor Protein by a Viral E3 Ligase Through Co-immunoprecipitation.

Co-immunoprecipitation is a technique widely utilized to isolate protein complexes and study protein-protein interactions. Ubiquitinated proteins could be identified by combining co-immunoprecipitation with SDS-PAGE followed by immunoblotting. In this chapter, we use Herpes Simplex Virus 1 immediate-early protein ICP0-mediated polyubiquitination of p50 as an example to describe the method to identify a ubiquitinated adaptor protein by a viral E3 ligase by co-immunoprecipitation.

New Pipeline for Analysing Fruit Proteolytic Products Used as Digestive Health Nutraceuticals.

Proteolytic products are extensively used in the nutraceutical sector to improve protein digestion and muscle quality in target populations (e.g., athletes or elderly). These products are processed using techniques that often lead to low purity but competitive pricing. Despite their widespread use and well-established production methods, the industry lacks standardized analytical methods for assessing these products and detecting potential fraud. This study proposes a comprehensive and harmonized pipeline for their analysis, which includes quantifying total soluble protein and proteolytic activity, as well as the determination of product stability and protein profile using SDS-PAGE and proteomic techniques. Despite the fact that protease extracts from pineapple had the highest protein content, most of the bromelain remained inactive, unlike in kiwi and papaya. SDS-PAGE revealed partial protein degradation of pineapple extracts, whereas kiwi extracts reflected a lower purification level but a higher protein integrity. The application of proteomic approaches strengthened the identification and origin tracing of the proteases. This study contributes to the development of a robust framework for analyzing proteolytic extracts, spanning from soluble protein quantification to protein profiling and activity determination. It may also ensure reliable supplier selection, high-quality manufacturing practices, and the implementation of optimal storage and formulation strategies in the nutraceutical industry.

Revisiting Protein Reversed-Phase Chromatography for Bottom-Up Proteomics.

We revisited protein reversed-phase chromatography (RP), using state-of-the-art RP columns developed for biopharmaceuticals, such as monoclonal antibodies, in order to evaluate the suitability of this methodology as a prefractionation step for bottom-up proteomics. The protein RP prefractionation (Prot-RP) method was compared with two other widely used prefractionation methods, SDS-PAGE and high-pH peptide RP (Pept-RP) by using cell lysates as samples. The overlap between fractions of Prot-RP was comparable to that of SDS-PAGE, and the protein recovery was approximately 2-fold higher. On the other hand, the overlap between fractions of Prot-RP was slightly larger than that of Pept-RP, but Prot-RP was able to identify more protein termini and more isoform-specific peptides than Pept-RP. Our results indicate that the combination of highly efficient protein prefractionation with modern mass spectrometers is particularly effective for proteoform profiling from cellular samples.

Polysaccharides Analysis of Cell Wall Using Carbohydrate Gel Electrophoresis.

The plant cell wall is rich in polysaccharides with high heterogeneity. Investigating the composition and structure of cell wall polysaccharides is crucial for understanding the functionalities of plant cell walls. Carbohydrate electrophoresis is a sensitive and rapid method to analyze polysaccharides qualitatively and quantitatively. The process includes digesting the polysaccharides with appropriate cleavage enzymes, labeling the reducing ends of the released oligosaccharides with a highly charged fluorophore, and separating the labeled oligosaccharides in a polyacrylamide gel via high-voltage electrophoresis. The generated fluorescence can be calculated as compared to that of oligosaccharide standards. Therefore, this is a convenient method for polysaccharide characterization that can be performed in most laboratories. Here, we introduce the detailed operational steps and precautions, which are helpful for researchers to quickly obtain the structural information of polysaccharides.

Combining SDS-PAGE to capillary zone electrophoresis-tandem mass spectrometry for high-resolution top-down proteomics analysis of intact histone proteoforms.

Mass spectrometry (MS)-based top-down proteomics (TDP) analysis of histone proteoforms provides critical information about combinatorial post-translational modifications (PTMs), which is vital for pursuing a better understanding of epigenetic regulation of gene expression. It requires high-resolution separations of histone proteoforms before MS and tandem MS (MS/MS) analysis. In this work, for the first time, we combined SDS-PAGE-based protein fractionation (passively eluting proteins from polyacrylamide gels as intact species for mass spectrometry, PEPPI-MS) with capillary zone electrophoresis (CZE)-MS/MS for high-resolution characterization of histone proteoforms. We systematically studied the histone proteoform extraction from SDS-PAGE gel and follow-up cleanup as well as CZE-MS/MS, to determine an optimal procedure. The optimal procedure showed reproducible and high-resolution separation and characterization of histone proteoforms. SDS-PAGE separated histone proteins (H1, H2, H3, and H4) based on their molecular weight and CZE provided additional separations of proteoforms of each histone protein based on their electrophoretic mobility, which was affected by PTMs, for example, acetylation and phosphorylation. Using the technique, we identified over 200 histone proteoforms from a commercial calf thymus histone sample with good reproducibility. The orthogonal and high-resolution separations of SDS-PAGE and CZE made our technique attractive for the delineation of histone proteoforms extracted from complex biological systems.

Sequential Double Immunoblotting with Peptide Antibodies.

Immunoblotting, also termed western blotting, is a powerful method for detection and characterization of proteins separated by various electrophoretic techniques. The combination of sodium dodecyl sulfate-poly acrylamide gel electrophoresis (SDS-PAGE), having high separating power, immunoblotting to synthetic membranes, and detection with highly specific peptide antibodies, is especially useful for studying individual proteins in relation to cellular processes, disease mechanisms, etc. Here, we describe a protocol for the sequential detection of various forms of an individual protein using peptide antibodies, exemplified by the characterization of antibody specificity for different forms of the protein calreticulin by double SDS-PAGE immunoblotting.

Detection and quantification of C-terminally tagged proteins by in-gel fluorescence.

The analysis of recombinant proteins in complex solutions is often accomplished with tag-specific antibodies in western blots. Recently, I introduced an antibody-free alternative wherein tagged proteins are visualized directly within polyacrylamide gels. For this, I used the protein ligase Connectase to selectively attach fluorophores to target proteins possessing an N-terminal recognition sequence. In this study, I extend this methodology to encompass the detection and quantification of C-terminally tagged proteins. Similar to the N-terminal labeling method, this adapted procedure offers increased speed, heightened sensitivity, and an improved signal-to-noise ratio when compared to western blots. It also eliminates the need for sample-specific optimization, enables more consistent and precise quantifications, and uses freely available reagents. This study broadens the applicability of in-gel fluorescence detection methods and thereby facilitates research on recombinant proteins.

Detection and Quantitation of Endogenous Membrane-Bound RAS Proteins and KRAS Mutants in Cancer Cell Lines Using 1D-SDS-PAGE LC-MS2.

In healthy cells, membrane-anchored wild-type RAS proteins (i.e., HRAS, KRAS4A, KRAS4B, and NRAS) regulate critical cellular processes (e.g., proliferation, differentiation, survival). When mutated, RAS proteins are principal oncogenic drivers in approximately 30% of all human cancers. Among them, KRAS mutants are found in nearly 80% of all patients diagnosed with RAS-driven malignancies and are regarded as high-priority anti-cancer drug targets. Due to the lack of highly qualified/specific RAS isoform and mutant RAS monoclonal antibodies, there is a vital need for an effective antibody-free approach capable of identifying and quantifying membrane-bound RAS proteins in isoform- and mutation-specific manner. Here, we describe the development of a simple antibody-free protocol that relies on ultracentrifugation to isolate the membrane fraction coupled with single-dimensional (1D) sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to fractionate and enrich membrane-bound endogenous RAS isoforms. Next, bottom-up proteomics that utilizes in-gel digestion followed by reversed-phase high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS2) is used for detection and relative quantitation of all wild-type RAS proteins (i.e., HRAS, KRAS4A, KRAS4B, and NRAS) and corresponding RAS mutants (e.g., G12D, G13D, G12S, G12V). Notably, this simple 1D-SDS-PAGE-HPLC-MS2-based protocol can be automated and widely applied to multiple cancer cell lines to investigate concentration changes in membrane-bound endogenous RAS proteins and corresponding mutants in the context of drug discovery.

Electro-elution-based purification of covalent DNA-protein cross-links.

Covalent DNA-protein cross-links (DPCs) are pervasive DNA lesions that challenge genome stability and can be induced by metabolic or chemotherapeutic cross-linking agents including reactive aldehydes, topoisomerase poisons and DNMT1 inhibitors. The purification of x-linked proteins (PxP), where DNA-cross-linked proteins are separated from soluble proteins via electro-elution, can be used to identify DPCs. Here we describe a versatile and sensitive strategy for PxP. Mammalian cells are collected following exposure to a DPC-inducing agent, embedded in low-melt agarose plugs and lysed under denaturing conditions. Following lysis, the soluble proteins are extracted from the agarose plug by electro-elution, while genomic DNA and cross-linked proteins are retained in the plug. The cross-linked proteins can then be analyzed by standard analytical techniques such as sodium dodecyl-sulfate-polyacrylamide gel electrophoresis followed by western blotting or fluorescent staining. Alternatively, quantitative mass spectrometry-based proteomics can be used for the unbiased identification of DPCs. The isolation and analysis of DPCs by PxP overcomes the limitations of alternative methods to analyze DPCs that rely on precipitation as the separating principle and can be performed by users trained in molecular or cell biology within 2-3 d. The protocol has been optimized to study DPC induction and repair in mammalian cells but may also be adapted to other sample types including bacteria, yeast and tissue samples.

Efficient experimental method for purifying allergens from aqueous extracts.

Studying the molecular and immunological basis of allergic diseases often requires purified native allergens. The methodologies for protein purification are usually difficult and may not be completely successful. The objective of this work was to describe a methodology to purify allergens from their natural source, while maintaining their native form. The purification strategy consists of a three-step protocol and was used for purifying five specific allergens, Ole e 1, Amb a 1, Alt a 1, Bet v 1 and Cup a 1. Total proteins were extracted in PBS (pH 7.2). Then, the target allergens were pre-purified and enriched by salting-out using increasing concentrations of ammonium sulfate. The allergens were further purified by anion exchange chromatography. Purification of Amb a 1 required an extra step of cation exchange chromatography. The detection of the allergens in the fractions obtained were screened by SDS-PAGE, and Western blot when needed. Further characterization of purified Amb a 1 was performed by mass spectrometry. Ole e 1, Alt a 1, Bet v 1 and Cup a 1 were obtained at > 90 % purity. Amb a 1 was obtained at > 85 % purity. Overall, we propose an easy-to-perform purification approach that allows obtaining highly pure allergens. Since it does not involve neither chaotropic nor organic reagents, we anticipate that the structural and biological functions of the purified molecule remain intact. This method provides a basis for native allergen purification that can be tailored according to specific needs.

A Simple and Rapid Microscale Method for Isolating Bacterial Lipopolysaccharides.

Bacterial endotoxins (lipopolysaccharides (LPSs)) are important mediators of inflammatory processes induced by Gram-negative microorganisms. LPSs are the key inducers of septic shock due to a Gram-negative bacterial infection; thus, the structure and functions of LPSs are of specific interest. Often, highly purified bacterial endotoxins must be isolated from small amounts of biological material. Each of the currently available methods for LPS extraction has certain limitations. Herein, we describe a rapid and simple microscale method for extracting LPSs. The method consists of the following steps: ultrasonic destruction of the bacterial material, LPS extraction via heating, LPS purification with organic solvents, and treatment with proteinase K. LPSs that were extracted by using this method contained less than 2-3% protein and 1% total nucleic acid. We also demonstrated the structural integrity of the O-antigen and lipid A via the sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) methods, respectively. We demonstrated the ability of the extracted LPSs to induce typical secretion of cytokines and chemokines by primary macrophages. Overall, this method may be used to isolate purified LPSs with preserved structures of both the O-antigen and lipid A and unchanged functional activity from small amounts of bacterial biomass.

Quality evaluation of highly purified human menopausal gonadotropin preparations by means of gel electrophoresis and mass spectrometry.

Human gonadotropins are glycoprotein hormones with a highly complex structure, which demands the application of sophisticated analytical methodologies to assess their quality. The principal objective of this study was a comparative evaluation of gel electrophoretic techniques and mass spectrometry-based methods for the quality study of the two urinary-derived, highly purified, human menopausal gonadotropin preparations, Menopur 75/75 I. U. and Meriofert 75 I. U. Molecular mass (Mr), isoelectric point (pI), and isoform pattern of studied compounds were estimated via SDS-PAGE and 2D gel electrophoresis, whereas matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was used for the downstream characterization of peptides obtained after in-gel tryptic digestion of selected protein spots. Additionally, for the estimation of the glycosylation pattern of these biologics, the enzymatic release of oligosaccharides was performed, and the isoform pattern was studied. Gel electrophoresis showed a typical electrophoretic behaviour for protein biotherapeutics medicines consisting of extremely complex spot patterns migrating at different masses and pIs. MS analysis proved to be a powerful tool for the identification and detailed characterization of the gonadotropins and the relevant peptides were identified with high sequence coverages. The results of this study are not only useful for the quality assessment of this class of complex biopharmaceuticals but may also serve as a supporting platform for further development of biopharmaceuticals based on modulation of the glycosylation pattern to enhance efficacy or reduce side effects.

Protocol for a semi-quantitative approach to identify protein S-palmitoylation in cultured cells by acyl biotin exchange assay.

Palmitoylation is a post-translational lipid modification in which palmitic acid is conjugated predominantly to cysteine residues of target proteins, allowing them to tether to cell membranes. Here, we describe a protocol to perform a stepwise acyl biotin exchange assay to identify protein S-palmitoylation. We describe steps for initial blocking of free thiols in protein lysates, subsequent replacement of thioester-linked palmitate groups with a biotin tag for affinity enrichment, and identification of palmitoylated proteins by SDS-PAGE. For complete details on the use and execution of this protocol, please refer to Leishman et al.1.

Microwave-assisted and methanol/acetic acid-free method for rapid staining of proteins in SDS-PAGE gels.

We describe a microwave-assisted, methanol and acetic acid-free, inexpensive method for rapid staining of SDS-PAGE proteins. Only citric acid, benzoic acid, and Coomassie brilliant blue G-250 (CBG) were used. Microwave irradiation reduced the detection duration, and proteins in a clear background were visualized within 30 min of destaining, after 2 min of fixing and 12 min of staining. By using this protocol, comparable band intensities were obtained to the conventional methanol/acetic acid method.

Rapid Separation and Display of Active Fibrinogenolytic Agents in Sipunculus nudus through Fibrinogen-Polyacrylamide Gel Electrophoresis.

Fibrinogenolytic agents that can dissolve fibrinogen directly have been widely used in anti-coagulation treatment. Generally, identifying new fibrinogenolytic agents requires the separation of each component first and then checking their fibrinogenolytic activities. Currently, polyacrylamide gel electrophoresis (PAGE) and chromatography are mostly used in the separating stage. Meanwhile, the fibrinogen plate assay and reaction products based PAGE are usually adopted to display their fibrinogenolytic activities. However, because of the spatiotemporal separation of those two stages, it is impossible to separate and display the active fibrinogenolytic agents with the same gel. To simplify the separating and displaying processes of fibrinogenolytic agent identification, we constructed a new fibrinogen-PAGE method to rapidly separate and display the fibrinogenolytic agents of peanut worms (Sipunculus nudus) in this study. This method includes fibrinogen-PAGE preparation, electrophoresis, renaturation, incubation, staining, and decolorization. The fibrinogenolytic activity and molecular weight of the protein can be detected simultaneously. According to this method, we successfully detected more than one active fibrinogenolytic agent of peanut wormhomogenate within 6 h. Moreover, this fibrinogen-PAGE method is time and cost-friendly. Furthermore, this method could be used to study the fibrinogenolytic agents of the other organisms.

A Practical Guide for the Quality Evaluation of Fluobodies/Chromobodies.

BACKGROUND: Fluorescent proteins (FPs) are pivotal reagents for flow cytometry analysis or fluorescent microscopy. A new generation of immunoreagents (fluobodies/chromobodies) has been developed by fusing recombinant nanobodies to FPs. METHODS: We analyzed the quality of such biomolecules by a combination of gel filtration and SDS-PAGE to identify artefacts due to aggregation or material degradation. RESULTS: In the SDS-PAGE run, unexpected bands corresponding to separate fluobodies were evidenced and characterized as either degradation products or artefacts that systematically resulted in the presence of specific FPs and some experimental conditions. The elimination of N-terminal methionine from FPs did not impair the appearance of FP fragments, whereas the stability and migration characteristics of some FP constructs were strongly affected by heating in loading buffer, which is a step samples undergo before electrophoretic separation. CONCLUSIONS: In this work, we provide explanations for some odd results observed during the quality control of fluobodies and summarize practical suggestions for the choice of the most convenient FPs to fuse to antibody fragments.

A thiol-anchored solvatochromic and fluorogenic molecular rotor for covalent protein labeling in SDS-PAGE and mitochondria specific fluorescence imaging.

Fluorescent labeling is a widely used method for protein detection and fluorescence imaging. A solvatochromic and fluorogenic molecular rotor DASPBCl was developed for covalent protein labeling in solution and SDS-PAGE, and also for stable mitochondria labeling and fluorescence imaging. The dye DASPBCl consisted of a 4-(N,N-dimethylamino)phenyl moiety as the electron donor and a positively charged N-benzylpyridinium moiety as the electron acceptor. A benzyl chloride group was introduced into the pyridine moiety for covalent labeling of thiol in proteins. When the fluorescent dye DASPBCl is covalently labeled to the thiol of proteins, significantly enhanced fluorescence was obtained, which is attributed to the polarity sensitivity caused solvatochromic effect from the hydrophobic protein structure and the viscosity sensitivity caused fluorogenic effect from the restriction of single bond rotation. DASPBCl exhibits high sensitivity and good linear response for protein detection in SDS-PAGE analysis with both the pre-staining method and post-staining method. DASPBCl was also successfully used for covalently protein-anchored fluorescence imaging of mitochondria in living cells.

Residual protein analysis by SDS-PAGE in clinically manufactured BM-MSC products.

Residual substances that are considered hazardous to the recipient must be removed from final cellular therapeutic products manufactured for clinical purposes. In doing so, quality rules determined by competent authorities (CAs) for the clinical use of tissue- and cell-based products can be met. In our study, we carried out residual substance analyses, and purity determination studies of trypsin and trypsin inhibitor in clinically manufactured bone marrow-derived mesenchymal stromal/stem cell products, using the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) method. Despite being a semiquantitative method, SDS-PAGE has several benefits over other methods for protein analysis, such as simplicity, convenience of use, and affordability. Due to its convenience and adaptability, SDS-PAGE is still a commonly used method in many laboratories, despite its limits in dynamic range and quantitative precision. Our goal in this work was to show that SDS-PAGE may be used effectively for protein measurement, especially where practicality and affordability are the major factors. The results of our study suggest a validated method to guide tissue and cell manufacturing sites for making use of an agreeable, accessible, and cost-effective method for residual substance analyses in clinically manufactured cellular therapies.

Protocol for isolating small cytosolic dsDNA from cultured murine cells.

We recently identified a class of small cytosolic double-stranded DNA (scDNA) approximately 20-40 bp in size in human and mouse cells. Here, we present a protocol for scDNA isolation from cultured murine cells. We describe steps for cytosolic compartment separation, DNA isolation in the cytosolic fraction using phenol-chloroform extraction, and ethanol precipitation. We then detail procedures for denaturing purified cytosolic DNA through urea polyacrylamide gel electrophoresis and obtaining scDNA in the cytosolic DNA fraction via gel purification. For complete details on the use and execution of this protocol, please refer to Liu et al.1.

Chemical cross-linking to study protein self-assembly in cellulo.

Many proteins self-assemble into dimers and higher-order oligomers. Therefore, the goal of this protocol is to characterize the conformational states of an endogenous protein of interest. Here, we present a protocol for assessing protein self-assembly in cell lysates using chemical cross-linking. We describe steps for chemical cross-linking with recombinant proteins as well as steps for cell culture and cell lysate preparation, chemical cross-linking, SDS-PAGE, and western blotting for the detection of endogenous proteins. For complete details on the use and execution of this protocol, please refer to Balaji et al.1.