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1.
Bioconjug Chem ; 35(2): 154-163, 2024 Feb 21.
Article En | MEDLINE | ID: mdl-38320084

Conventional techniques for purifying macromolecular conjugates often require complex and costly installments that are inaccessible to most laboratories. In this work, we develop a one-step micropreparative method based on a trilayered polyacrylamide gel electrophoresis (MP-PAGE) setup to purify biological samples, synthetic nanoparticles, as well as biohybrid complexes. We apply this method to recover DNA from a ladder mixture with yields of up to 90%, compared to the 58% yield obtained using the conventional crush-and-soak method. MP-PAGE was also able to isolate enhanced yellow fluorescence protein (EYFP) from crude cell extract with 90% purity, which is comparable to purities achieved through a more complex two-step purification procedure involving size exclusion and immobilized metal-ion affinity chromatography. This technique was further extended to demonstrate size-dependent separation of a commercial mixture of graphene quantum dots (GQDs) into three different fractions with distinct optical properties. Finally, MP-PAGE was used to isolate DNA-EYFP and DNA-GQD bioconjugates from their reaction mixture of DNA and EYFP and GQD precursors, samples that otherwise could not be effectively purified by conventional chromatography. MP-PAGE thus offers a rapid and versatile means of purifying biological and synthetic nanomaterials without the need for specialized equipment.


Proteins , Quantum Dots , Electrophoresis, Polyacrylamide Gel , Quantum Dots/chemistry , Chromatography, Affinity , DNA
2.
Methods Mol Biol ; 2763: 79-97, 2024.
Article En | MEDLINE | ID: mdl-38347402

Distinct bands of mucins cannot be banded using a gel electrophoresis based on a molecular sieving effect due to their very large molecular weight and remarkable diversity in glycosylation. In contrast, membrane electrophoresis can separate mucins as round bands. Here, we present an analysis of mucin separation via membrane electrophoresis using a porous polyvinylidene difluoride membrane, which is highly stable against chemical modifications and various organic solvents. The separated mucins can not only be stained with dyes but also with antibodies and lectins, and glycans can be released from the excised bands and analyzed.


Coloring Agents , Mucins , Electrophoresis/methods , Mucins/chemistry , Coloring Agents/chemistry , Lectins , Glycosylation , Electrophoresis, Polyacrylamide Gel
3.
Anal Chim Acta ; 1291: 342219, 2024 Feb 22.
Article En | MEDLINE | ID: mdl-38280790

The detection of intrinsic protein fluorescence is a powerful tool for studying proteins in their native state. Thanks to its label-free and stain-free feature, intrinsic fluorescence detection has been introduced to polyacrylamide gel electrophoresis (PAGE), a fundamental and ubiquitous protein analysis technique, to avoid the tedious detection process. However, the reported methods of intrinsic fluorescence detection were incompatible with online PAGE detection or standard slab gel. Here, we fulfilled online intrinsic fluorescence imaging (IFI) of the standard slab gel to develop a PAGE-IFI method for real-time and quantitative protein detection. To do so, we comprehensively investigated the arrangement of the deep-UV light source to obtain a large imaging area compatible with the standard slab gel, and then designed a semi-open gel electrophoresis apparatus (GEA) to scaffold the gel for the online UV irradiation and IFI with low background noise. Thus, we achieved real-time monitoring of the protein migration, which enabled us to determine the optimal endpoint of PAGE run to improve the sensitivity of IFI. Moreover, online IFI circumvented the broadening of protein bands to enhance the separation resolution. Because of the low background noise and the optimized endpoint, we showcased the quantitative detection of bovine serum albumin (BSA) with a limit of detection (LOD) of 20 ng. The standard slab gel provided a high sample loading volume that allowed us to attain a wide linear range of 0.03-10 µg. These results indicate that the PAGE-IFI method can be a promising alternative to conventional PAGE and can be widely used in molecular biology labs.


Optical Imaging , Serum Albumin, Bovine , Electrophoresis, Polyacrylamide Gel
4.
Appl Microbiol Biotechnol ; 108(1): 176, 2024 Jan 26.
Article En | MEDLINE | ID: mdl-38277014

The demand for massive quantities of therapeutic active antimicrobial peptides (AMPs) is high due to their potential as alternatives to antibiotics. However, each antimicrobial peptide has unique properties, necessitating distinct synthesis and purification strategies for their large-scale production. In this study, we bio-synthesized and purified a functional enhanced variant of the AMP epinecidin-1, known as Ac-Var-1 (acid-cleavable variant-1). To generate the active peptide, we cloned the gene for Ac-Var-1 with acid-cleavable site (aspartic acid-proline) into the pET-32a expression vector, purified the fusion protein by His tag enrichment chromatography, and performed acid cleavage to release the active Ac-Var-1 peptide. After acid cleavage, the active Ac-Var-1 was purified and characterized by SDS-PAGE and mass spectrometry. The results from both techniques provided confirmation of the intactness of the purified Ac-Var-1. The Ac-Var-1 inhibited the growth of pathogenic Escherichia coli and Staphylococcus aureus. KEY POINTS : • Epinecidin-1 is a well-known antimicrobial peptide having multipotential bioactivities. • Epinecidin-1 variant is developed via the site-directed mutagenesis method to improve its structural stability and bioactivity. • AC-Var-1 development is an economical and easy method to remove peptide from tag protein.


Antimicrobial Cationic Peptides , Staphylococcal Infections , Humans , Antimicrobial Cationic Peptides/genetics , Antimicrobial Cationic Peptides/pharmacology , Antimicrobial Cationic Peptides/chemistry , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Electrophoresis, Polyacrylamide Gel , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism
5.
Article En | MEDLINE | ID: mdl-38181709

Recombinant factor VII, produced in recombinant BHK cell line, is secreted as a single chain zymogen form (rFVII, non-activated) in cell culture supernatant and subsequently converts to its active form during anion exchange chromatography step in the downstream purification process, with the aid of calcium ion. Single chain rFVII impurity (non-activated form) in final drug products should not exceed more than 3.0 % of total rFVIIa content. Therefore, one of the most essential quality control tests in pharmaceutical companies is to precisely quantify and report this impurity. SDS-PAGE, as a traditional method in quality control laboratories to quantify single chain rFVII, is a laborious, time-consuming, low output, and semi-quantitative method for quantification of non-activated form impurity which utilizes a densitometer to scan the gel and calculate the non-activated form band density. In this work, we developed two novel instrumental-based techniques (SE-UPLC and CE-SDS) with superior precision, accuracy, sensitivity, and efficiency that overcome SDS-PAGE shortcomings. The results of both methods were comparable to SDS-PAGE and showed an even higher correlation with expected values. Finally, we concluded that these two methods could be used as a high throughput routine method in quality control laboratories as an alternative choice to manual SDS-PAGE.


Chromatography, High Pressure Liquid , Cell Line , Electrophoresis, Polyacrylamide Gel
6.
J Agric Food Chem ; 72(6): 3099-3112, 2024 Feb 14.
Article En | MEDLINE | ID: mdl-38291573

Among fruits susceptible to enzymatic browning, olive polyphenol oxidase (OePPO) stood out as being unisolated from a natural source until this study, wherein we successfully purified and characterized the enzyme. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of heated and nonheated OePPO revealed distinct molecular weights of 35 and 54 kDa, respectively, indicative of its oligomeric nature comprising active and C-terminal subunits. OePPO displayed latency, fully activating with 5 mM SDS under optimal conditions of pH 7.5 and 15 °C. The enzyme demonstrated monophenolase activity and showcased the highest efficiency toward hydroxytyrosol. Despite its low optimal temperature, OePPO exhibited high thermal resistance, maintaining stability up to 90 °C. However, beyond this threshold, the oligomeric enzyme disassociated, yielding a denatured main subunit and C-terminal fragments. Six OePPO genes were found in the fruits. Tryptic digestion identified the enzyme as mature OePPO1 (INSDC OY733096), while mass spectrometry detected the active form mass alongside several C-terminal fragments, revealing potential cleavage sites (Gly407, Tyr408).


Olea , Catechol Oxidase/genetics , Catechol Oxidase/chemistry , Hot Temperature , Electrophoresis, Polyacrylamide Gel
7.
RNA ; 30(3): 298-307, 2024 Feb 16.
Article En | MEDLINE | ID: mdl-38164606

Several methods are available to visualize and assess the kinetics and efficiency of elemental steps of protein biosynthesis. However, each of these methods has its own limitations. Here, we present a novel, simple and convenient tool for monitoring stepwise in vitro translation initiated by BODIPY-Met-tRNA. Synthesis and release of very short, 1-7 amino acids, BODIPY-labeled peptides, can be monitored using urea-polyacrylamide gel electrophoresis. Very short BODIPY-labeled oligopeptides might be resolved this way, in contrast to widely used Tris-tricine gel electrophoresis, which is suitable to separate peptides larger than 1 kDa. The method described in this manuscript allows one to monitor the steps of translation initiation, peptide transfer, translocation, and termination as well as their inhibition at an unprecedented single amino acid resolution.


Boron Compounds , Peptides , RNA, Transfer, Amino Acyl , RNA, Transfer, Amino Acyl/chemistry , Peptides/metabolism , RNA, Transfer/metabolism , Electrophoresis, Polyacrylamide Gel , Protein Biosynthesis
8.
Anal Biochem ; 684: 115373, 2024 01 01.
Article En | MEDLINE | ID: mdl-37926185

Agarose gel electrophoresis is performed routinely by molecular biologists as both an analytical and a preparative method for characterization of nucleic acids. Gel analysis of highly dilute DNA solutions is challenging because of the limited sensitivity of detection available with conventional methods. In this study a new approach is described for concentrating samples directly within gels called SURE (successive reloading) electrophoresis. The approach involves loading of dilute samples multiple times into a single well, with each loading followed by a brief pulse of electrical current before the next sample is loaded. The procedure generates single bands created by molecular stacking that exhibit strongly enhanced signal intensities and minimal band broadening. Using optimized voltages and time intervals as many as 20 successive loadings could be performed and up to 800 µL could be loaded into a single well. Gel extraction and fluorescent quantitation demonstrated that approximately 97 % of the DNA from each loading was incorporated into the resultant band. Highly dilute DNA samples (<0.0007 ng per microliter) could be readily detected after six loadings. The method produced good results with either TAE or TBE as electrophoresis buffers, using loading dyes with or without SDS, and in both minigels and large gels.


DNA , Nucleic Acids , Electrophoresis, Agar Gel/methods , Gels , Electrophoresis, Polyacrylamide Gel
9.
Front Immunol ; 14: 1279612, 2023.
Article En | MEDLINE | ID: mdl-37954579

Factor I (FI) is an essential regulator of the complement system. Together with co-factors, FI degrades C3b, which inhibits further complement activation. Genetic mutations in FI are associated with pathological conditions like age-related macular degeneration and atypical hemolytic uremic syndome. Here, we evaluated eight recombinant FI genetic variants found in patients. We assessed FI's co-factor activity in the presence of two co-factors; Factor H and soluble CR1. Different analytical assays were employed; SDS-PAGE to evaluate the degradation of C3b, ELISA to measure the generation of fluid phase iC3b and the degradation of surface-bound C3b using a novel Luminex bead-based assay. We demonstrate that mutations in the FIMAC and SP domains of FI led to significantly reduced protease activity, whereas the two analyzed mutations in the LDLRA2 domain did not result in any profound changes in FI's function. The different assays employed displayed a strong positive correlation, but differences in the activity of the genetic variants Ile55Phe and Gly261Asp could only be observed by combining different methods and co-factors for evaluating FI activity. In conclusion, our results provide a new perspective regarding available diagnostic tools for assessing the impact of mutations in FI.


Complement C3b , Complement Factor I , Humans , Complement Factor I/genetics , Complement Factor I/metabolism , Complement C3b/metabolism , Mutation , Enzyme-Linked Immunosorbent Assay , Electrophoresis, Polyacrylamide Gel
10.
Sci Rep ; 13(1): 19862, 2023 Nov 14.
Article En | MEDLINE | ID: mdl-37963965

Ferritin is a ubiquitous intracellular iron storage protein that plays a crucial role in iron homeostasis. Animal tissue ferritins consist of multiple isoforms (or isoferritins) with different proportions of H and L subunits that contribute to their structural and compositional heterogeneity, and thus physiological functions. Using size exclusion and anion exchange chromatography, capillary isoelectric focusing (cIEF), and SDS-capillary gel electrophoresis (SDS-CGE), we reveal for the first time a significant variation in ferritin subunit composition and isoelectric points, in both recombinant and native ferritins extracted from animal organs. Our results indicate that subunits composition is the main determinant of the mean pI of recombinant ferritin heteropolymers, and that ferritin microheterogeneity is a common property of both natural and recombinant proteins and appears to be an intrinsic feature of the cellular machinery during ferritin expression, regulation, post-translational modifications, and post-subunits assembly. The functional significance and physiological implications of ferritin heterogeneity in terms of iron metabolism, response to oxidative stress, tissue-specific functions, and pathological processes are discussed.


Ferritins , Iron , Animals , Ferritins/metabolism , Isoelectric Focusing , Electrophoresis, Polyacrylamide Gel , Iron/metabolism , Isoelectric Point
11.
Biochim Biophys Acta Gen Subj ; 1867(12): 130494, 2023 12.
Article En | MEDLINE | ID: mdl-37865174

NFE2L1 (also known as NRF1) is a member of the nuclear erythroid 2-like family of transcription factors and is critical for counteracting various types of cellular stress such as oxidative, proteotoxic or metabolic stress. This unique transcription factor is also known to undergo changes, including post-translational modifications, limited proteolysis or translocation into the nucleus, before it exerts full transcriptional activity. As a result, there are various molecular forms with distinct sizes for this protein, while the precise nature of each form remains elusive. In this study, the N-glycosylated status of NFE2L1 in cells was examined. The findings revealed that when NFE2L1 was deglycosylated by PNGase F, the size-shift on SDS-PAGE was minimal. This was in contrast to deglycosylation by Endo H, which resulted in a clear size-shift, even though N-linked GlcNAc residues remained on the protein. It was found that this unusual behavior of PNGase-deglycosylated NFE2L1 was dependent on the conversion of the glycosylated-Asn to Asp, resulting in the introduction of more negative charges into the core peptide of NFE2L1. We also demonstrate that NGLY1-mediated deglycosylation and DDI2-mediated proteolytic processing of NFE2L1 are not strictly ordered reactions. Our study will allow us to better understand the precise structures as well as biochemical properties of the various forms of NFE2L1.


Amino Acids , Transcription Factors , Amino Acids/metabolism , Transcription Factors/metabolism , Proteolysis , Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase/metabolism , Electrophoresis, Polyacrylamide Gel
12.
Int J Mol Sci ; 24(19)2023 Sep 28.
Article En | MEDLINE | ID: mdl-37834160

The identification of new genes/proteins involved in breast cancer (BC) occurrence is widely used to discover novel biomarkers and understand the molecular mechanisms of BC initiation and progression. The jumping translocation breakpoint (JTB) gene may act both as a tumor suppressor or oncogene in various types of tumors, including BC. Thus, the JTB protein could have the potential to be used as a biomarker in BC, but its neoplastic mechanisms still remain unknown or controversial. We previously analyzed the interacting partners of JTBhigh protein extracted from transfected MCF7 BC cell line using SDS-PAGE complemented with in-solution digestion, respectively. The previous results suggested the JTB contributed to the development of a more aggressive phenotype and behavior for the MCF7 BC cell line through synergistic upregulation of epithelial-mesenchymal transition (EMT), mitotic spindle, and fatty acid metabolism-related pathways. In this work, we aim to complement the previously reported JTB proteomics-based experiments by investigating differentially expressed proteins (DEPs) and tumorigenic pathways associated with JTB overexpression using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Statistically different gel spots were picked for protein digestion, followed by nanoliquid chromatography-tandem mass spectrometry (nLC-MS/MS) analysis. We identified six DEPs related to the JTBhigh condition vs. control that emphasize a pro-tumorigenic (PT) role. Twenty-one proteins, which are known to be usually overexpressed in cancer cells, emphasize an anti-tumorigenic (AT) role when low expression occurs. According to our previous results, proteins that have a PT role are mainly involved in the activation of the EMT process. Interestingly, JTB overexpression has been correlated here with a plethora of significant upregulated and downregulated proteins that sustain JTB tumor suppressive functions. Our present and previous results sustain the necessity of the complementary use of different proteomics-based methods (SDS-PAGE, 2D-PAGE, and in-solution digestion) followed by tandem mass spectrometry to avoid their limitations, with each method leading to the delineation of specific clusters of DEPs that may be merged for a better understanding of molecular pathways and neoplastic mechanisms related to the JTB's role in BC initiation and progression.


Breast Neoplasms , Tandem Mass Spectrometry , Humans , Female , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , MCF-7 Cells , Carcinogenesis , Electrophoresis, Polyacrylamide Gel , Chromatography , Electrophoresis, Gel, Two-Dimensional
13.
J Vis Exp ; (199)2023 09 22.
Article En | MEDLINE | ID: mdl-37811931

The technique described here can be used to identify specific myosin heavy chain (MHC) isoforms in segments of individual muscle fibers using dot blotting, hereafter referred to as Myosin heavy chain detection by Dot Blotting for IDentification of muscle fiber type (MyDoBID). This protocol describes the process of freeze-drying human skeletal muscle and isolating segments of single muscle fibers. Using MyDoBID, type I and II fibers are classified with MHCI- and IIa-specific antibodies, respectively. Classified fibers are then combined into fiber type-specific samples for each biopsy. The total protein in each sample is determined by Sodium Dodecyl-Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) and UV-activated gel technology. The fiber type of samples is validated using western blotting. The importance of performing protein loading normalization to enhance target protein detection across multiple western blots is also described. The benefits of consolidating classified fibers into fiber type-specific samples compared to single-fiber western blots, include sample versatility, increased sample throughput, shorter time investment, and cost-saving measures, all while retaining valuable fiber type-specific information that is frequently overlooked using homogenized muscle samples. The purpose of the protocol is to achieve accurate and efficient identification of type I and type II fibers isolated from freeze-dried human skeletal muscle samples. These individual fibers are subsequently combined to create type I and type II fiber type-specific samples. Furthermore, the protocol is extended to include the identification of type IIx fibers, using Actin as a marker for fibers that were negative for MHCI and MHCIIa, which are confirmed as IIx fibers by western blotting. Each fiber type-specific sample is then used to quantify the expression of various target proteins using western blotting techniques.


Muscle, Skeletal , Myosin Heavy Chains , Humans , Myosin Heavy Chains/metabolism , Muscle, Skeletal/physiology , Muscle Fibers, Skeletal/metabolism , Blotting, Western , Protein Isoforms/metabolism , Electrophoresis, Polyacrylamide Gel
14.
Anal Chem ; 95(37): 14009-14015, 2023 09 19.
Article En | MEDLINE | ID: mdl-37672655

Protein mass spectrometry imaging (MSI) with electrospray-based ambient ionization techniques, such as nanospray desorption electrospray ionization (nano-DESI), generates data sets in which each pixel corresponds to a mass spectrum populated by peaks corresponding to multiply charged protein ions. Importantly, the signal associated with each protein is split among multiple charge states. These peaks can be transformed into the mass domain by spectral deconvolution. When proteins are imaged under native/non-denaturing conditions to retain non-covalent interactions, deconvolution is particularly valuable in helping interpret the data. To improve the acquisition speed, signal-to-noise ratio, and sensitivity, native MSI is usually performed using mass resolving powers that do not provide isotopic resolution, and conventional algorithms for deconvolution of lower-resolution data are not suitable for these large data sets. UniDec was originally developed to enable rapid deconvolution of complex protein mass spectra. Here, we developed an updated feature set harnessing the high-throughput module, MetaUniDec, to deconvolve each pixel of native MSI data sets and transform m/z-domain image files to the mass domain. New tools enable the reading, processing, and output of open format .imzML files for downstream analysis. Transformation of data into the mass domain also provides greater accessibility, with mass information readily interpretable by users of established protein biology tools such as sodium dodecyl sulfate polyacrylamide gel electrophoresis.


Algorithms , Diagnostic Imaging , Electrophoresis, Polyacrylamide Gel , Mass Spectrometry , Signal-To-Noise Ratio
15.
Biochemistry (Mosc) ; 88(6): 731-740, 2023 Jun.
Article En | MEDLINE | ID: mdl-37748870

Endopeptidases IdeS and IdeZ (streptococcal virulence factors that specifically cleave IgG heavy chains) are of particular interest because of their potential use in biotechnology, medicine, and veterinary. Genes encoding these enzymes were cloned and expressed in Escherichia coli heterologous expression system (ideS was cloned from a Streptococcus pyogenes collection strain; ideZ from Streptococcus zooepidemicus was synthesized). The 6His-tag was introduced into the amino acid sequence of each endopeptidase, and IdeS and IdeZ were purified by metal affinity chromatography to an apparent homogeneity (according to polyacrylamide gel electrophoresis). Purified enzymes were active against human and animal IgGs; their specificity toward human IgGs was confirmed by polyacrylamide gel electrophoresis. Recombinant IdeZ was used for immunological analysis of equine strangles infection (diagnostics and determination of the titer of specific antibodies in blood). Hence, IdeZ can be used in veterinary and sanitary microbiology to diagnose infections caused by Streptococcus equi and S. zooepidemicus in addition to its application in medicine and biotechnology.


Endopeptidases , Insulysin , Humans , Animals , Horses , Endopeptidases/genetics , Amino Acid Sequence , Biotechnology , Chromatography, Affinity , Electrophoresis, Polyacrylamide Gel , Escherichia coli/genetics , Immunosuppressive Agents
16.
Food Res Int ; 172: 113180, 2023 10.
Article En | MEDLINE | ID: mdl-37689931

Roasting could modify the protein structure/conformation, contributing to changes in functional properties. Here we investigated the effects of pre-roasting on the extraction efficiency, structural and functional properties of pea protein concentrates and isolates (PPC and PPI) produced from yellow split peas. The shorter roasting times (150 °C, 10 and 20 min) had little effect on protein yields and could increase the solubility of PPC or PPI by âˆ¼ 12% at pH 7 and enhance the solubility of PPI by âˆ¼ 12% (10-min roasting) and âˆ¼ 24% (20-min roasting) at pH 3. However, a longer duration of pre-roasting (150 °C, 30 min) significantly reduced the extraction efficiency of PPC and PPI by âˆ¼ 30% and âˆ¼ 61%, respectively. Meanwhile, pre-roasting had minor effects on SDS-PAGE profiles and the secondary structures of pea proteins but significantly altered tertiary structures by reducing free sulfhydryl groups, increasing disulfide bonds and surface hydrophobicity. As for the emulsifying properties, pre-roasting improved the emulsion ability index (EAI) of PPC and PPI but decreased the emulsion stability index (ESI) of PPC and had no significant effect on PPI. Moreover, PPC and PPI with shorter pre-roasting duration (10 and 20 min) had endothermic peaks and showed a slight decrease in the denaturation temperature (Td) and the onset temperature (To), respectively. Overall, the study demonstrated that controlled pre-roasting at 150 °C for 10 min and 20 min altered protein structures (mainly tertiary structures), improving the solubility and EAI of pea proteins at pH 7, while retaining their thermal properties in comparison to unroasted samples.


Lathyrus , Pea Proteins , Emulsions , Protein Conformation , Electrophoresis, Polyacrylamide Gel
17.
Methods ; 219: 30-38, 2023 11.
Article En | MEDLINE | ID: mdl-37690737

The development of compounds that can selectively bind with non-canonical DNA structures has expanded in recent years. Junction DNA, including three-way junctions (3WJs) and four-way Holliday junctions (HJs), offer an intriguing target for developmental therapeutics as both 3WJs and HJs are involved in DNA replication and repair processes. However, there are a limited number of assays available for the analysis of junction DNA binding. Here, we describe the design and execution of multiplex fluorescent polyacrylamide gel electrophoresis (PAGE) and microscale thermophoresis (MST) assays that enable evaluation of junction-binding compounds. Two well characterised junction-binding compounds-a C6 linked bis-acridine ligand and an iron(II)-bound peptide helicate, which recognise HJs and 3WJs, respectively-were employed as probes for both MST and PAGE experiments. The multiplex PAGE assay expands beyond previously reported fluorescent PAGE as it uses four individual fluorophores that can be combined to visualise single-strands, pseudo-duplexes, and junction DNA present during 3WJ and HJ formation. The use of MST to identify the binding affinity of junction binding agents is, to our knowledge, first reported example of this technique. The combined use of PAGE and MST provides complementary results for the visualisation of 3WJ and HJ formation and the direct binding affinity (Kd and EC50) of these agents. These assays can be used to aid the discovery and design of new therapeutics targeting non-canonical nucleic acid structures.


DNA, Cruciform , DNA , DNA/chemistry , DNA Replication , Electrophoresis, Polyacrylamide Gel
18.
Molecules ; 28(17)2023 Aug 24.
Article En | MEDLINE | ID: mdl-37687048

The complete mechanism behind starch regulation has not been fully characterized. However, significant progress can be achieved through proteomic approaches. In this work, we aimed to characterize the starch-interacting proteins in potato (Solanum tuberosum L. cv. Desiree) tubers under variable circumstances. Starch-interacting proteins were extracted from developing tubers of wild type and transgenic lines containing antisense inhibition of glucan phosphorylases. Further, proteins were separated by SDS-PAGE and characterized through mass spectrometry. Additionally, starch-interacting proteins were analyzed in potato tubers stored at different temperatures. Most of the proteins strongly interacting with the potato starch granules corresponded to proteins involved in starch metabolism. GWD and PWD, two dikinases associated with starch degradation, were consistently found bound to the starch granules. This indicates that their activity is not only restricted to degradation but is also essential during storage starch synthesis. We confirmed the presence of protease inhibitors interacting with the potato starch surface as previously revealed by other authors. Starch interacting protein profiles of transgenic tubers appeared differently from wild type when tubers were stored under different temperatures, indicating a differential expression in response to changing environmental conditions.


Solanum tuberosum , Animals , Solanum tuberosum/genetics , Proteomics , Animals, Genetically Modified , Electrophoresis, Polyacrylamide Gel , Starch
19.
J Agric Food Chem ; 71(37): 13920-13933, 2023 Sep 20.
Article En | MEDLINE | ID: mdl-37688549

In this study, changes in the physical, structural, and assembly characteristics of silver carp myofibrillar proteins (MPs) at different ionic strength (I) values were investigated. Moreover, the differential proteomic profile of soluble MPs was analyzed using 4D proteomics based on timsTOF Pro mass spectrometry. Solubility of MPs significantly increased at high I (>0.3), and the increase in I enhanced the apparent viscosity, fluorescence intensity, surface hydrophobicity, and α-helix content of MPs solution. Particle size and sodium dodecyl sulfate-polyacrylamide gel electrophoresis patterns also supported the solubility profiles. Transmission electron microscopy and atomic force microscopy observations revealed the morphological assembly and disassembly of MPs under different I conditions. Finally, proteomic analysis revealed the evolution law of salt-induced solubilization of MPs and the critical molecular characteristics in different I environments. The number of differentially abundant proteins (DAPs) decreased with the increase of I, and most DAPs related to the muscle filament sliding, contraction and assembly, actinin binding, and actin filament binding. The soluble abundance of myosin and some structural proteins was dependent on I, and structural proteins in the Z-disk and M-band might contribute to the solubilization of myosin. Our findings provide insightful information about the impact of common I on the solubility pattern of MPs from freshwater fish.


Carps , Proteomics , Animals , Electrophoresis, Polyacrylamide Gel , Fresh Water , Mass Spectrometry
20.
Protein Sci ; 32(10): e4771, 2023 10.
Article En | MEDLINE | ID: mdl-37638851

Serum autoantibody profiles are unique to individuals and reflect the level and history of autoimmunity and tumor immunity. The identification of autoantibody biomarkers is critical for the development of immune monitoring systems for immune-related disorders. Here, we present a practical method for large-scale autoantibody discovery using total cellular proteins from cultured mammalian cells. We found that nucleic acid-free and fully denatured water-soluble total cellular proteins from mammalian cells were superior, allowing precise separation by reversed-phase HPLC after preparing a large set of homogeneous total cellular proteins. After separating the proteins based on hydrophobicity, the fractionated samples were subjected to molecular mass analysis using conventional SDS-PAGE. The resulting two-dimensional gel electrophoresis was successfully employed for immune blotting and LC-MS/MS analysis. All procedures, including TRIzol-based total cellular protein extraction, solubilization of denatured proteins, reversed-phase HPLC separation, and SDS-PAGE, were highly reproducible and easily scalable. We propose this novel two-dimensional gel electrophoresis system as an alternative proteomics-based methodology suitable for large-scale autoantibody discovery.


Autoantibodies , Tandem Mass Spectrometry , Animals , Humans , Chromatography, Liquid , Proteins/analysis , Electrophoresis, Polyacrylamide Gel , Hydrophobic and Hydrophilic Interactions , Mammals
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