Gel- based proteomics is one of the most versatile methods for fractionating protein complexes. Among these methods, two dimensional- polyacrylamide gel electrophoresis (2-DE) represents a mainstay orthogonal approach, which is popularly used to simultaneously fractionate, identify, and quantify proteins when coupled with mass spectrometric identification or other immunological tests. Although 2-DE was first introduced more than three decades ago, several challenges and limitations to its utility still exist. This review discusses the principles of 2-DE as well as both recent methodological advances and new applications.

Mucopolysaccharidosis type I (MPS I) is due to deficiency of α-l-iduronidase (IDUA) and subsequent storage of undegraded glycosaminoglycans (GAG). The severe form of the disease, known as Hurler syndrome, is characterized by mental retardation and neurodegeneration of unknown etiology. To identify potential biomarkers and unveil the neuropathology mechanism of MPS I disease, two-dimensional polyacrylamide gel electrophoresis (PAGE) and nanoliquid chromatography-tandem mass spectrometry (nanoLC-MS/MS) were applied to compare proteome profiling of brains from MPS I and control mice (5-month old). A total of 2055 spots were compared, and 25 spots (corresponding to 50 different proteins) with a fold change ≥3.5 and a p value <0.05 between MPS I and control mice were further analyzed by nanoLC-MS/MS. These altered proteins could be divided into three major groups based on Gene Ontology (GO) terms: proteins involved in metabolism, neurotransmission and cytoskeleton. Cytoskeletal proteins including ACTA1, ACTN4, TUBB4B and DNM1 were significantly downregulated. STXBP1, a regulator of synaptic vesicle fusion and docking was also downregulated, indicating impaired synaptic transmission. Additionally, proteins regulating Ca2+ and H+ homeostasis including ATP6V1B2 and RYR3 were downregulated, which may be related to disrupted autophagic and endocytotic pathways. Notably, there is no altered expression in proteins associated with cell death, ubiquitin or inflammation. These results for the first time highlight the important role of alterations in metabolism pathways, intracellular ionic homeostasis and the cytoskeleton in the neuropathology of MPS I disease. The proteins identified in this study would provide potential biomarkers for diagnostic and therapeutic studies of MPS I.

Viperidae snakes are the most important agents of snakebites in Brazil. The protein composition of snake venoms has been frequently analyzed by means of electrophoretic techniques, but the interaction of proteins in venoms has barely been addressed. An electrophoretic technique that has gained prominence to study this type of interaction is blue native polyacrylamide gel electrophoresis (BN-PAGE), which allows for the high-resolution separation of proteins in their native form. These protein complexes can be further discriminated by a second-dimension gel electrophoresis (SDS-PAGE) from lanes cut from BN-PAGE. Once there is no study on the use of bidimensional BN/SDS-PAGE with snake venoms, this study initially standardized the BN/SDS-PAGE technique in order to evaluate protein interactions in Bothrops atrox, Bothrops erythromelas, and Bothrops jararaca snake venoms. Results of BN/SDS-PAGE showed that native protein complexes were present, and that snake venom metalloproteinases and venom serine proteinases maintained their enzymatic activity after BN/SDS-PAGE. C-type lectin-like proteins were identified by Western blotting. Therefore, bidimensional BN/SDS-PAGE proved to be an easy, practical, and efficient method for separating functional venom proteins according to their assemblage in complexes, as well as to analyze their biological activities in further details.

In the last decades, prevalence of autism spectrum disorder (ASD) has been on the rise. However, clear aetiology is still elusive and improvements in early diagnosis are needed. To uncover possible biomarkers present in ASD, we used two-dimensional polyacrylamide gel electrophoresis and nanoliquid chromatography-tandem mass spectrometry (nanoLC-MS/MS), to compare salivary proteome profiling of children with ASD and controls. A total of 889 spots were compared and only those spots with a fold change ≥1.7 and a P-value <0.05 or a fold change of ≥3.0 between ASD cases and controls were analysed by nanoLC-MS/MS. Alpha-amylase, CREB-binding protein, p532, Transferrin, Zn alpha2 glycoprotein, Zymogen granule protein 16, cystatin D and plasminogen were down-regulated in ASD. Increased expression of proto-oncogene Frequently rearranged in advanced T-cell lymphomas 1 (FRAT1), Kinesin family member 14, Integrin alpha6 subunit, growth hormone regulated TBC protein 1, parotid secretory protein, Prolactin-inducible protein precursor, Mucin-16, Ca binding protein migration inhibitory factor-related protein 14 (MRP14) was observed in individuals with ASD. Many of the identified proteins have previously been linked to ASD or were proposed as risk factors of ASD at the genetic level. Some others are involved in pathological pathways implicated in ASD causality such as oxidative stress, lipid and cholesterol metabolism, immune system disturbances and inflammation. These data could contribute to protein signatures for ASD presence, risk and subtypes, and advance understanding of ASD cause as well as provide novel treatment targets for ASD.

In an effort to develop an analytical method capable of finding new metalloproteins, this is the first report of a new diagonal gel electrophoresis method to isolate and identify metalloproteins, based on the molecular recognition of holo- and apo-metalloproteins (metalbound and -free forms, respectively) by CBB G-250 dye and employing metal ion contaminant sweeping-blue native-polyacrylamide gel electrophoresis (MICS-BN-PAGE). The difference in electrophoretic mobilities between holo- and apo-forms was exaggerated as a result of interactions between the metalloproteins and the dye with no metal ion dissociation. The different binding modes of proteins with CBB G-250 dye, primarily related to hydrogen bonding, were confirmed by capillary zone electrophoresis (CZE) and molecular docking simulations. Due to in-gel holo/apo conversion between the first and second dimensions of PAGE, holo-metalloproteins in the original sample were completely isolated as spots off the diagonal line in the second dimension of PAGE. To prove the high efficiency of this method for metalloprotein analysis, we successfully identified a copper-binding protein from a total bacterial soluble extract for the first time.

Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) followed by mass spectrometric identification of the proteins in the protein spots has become a central tool in proteomics. CMT167(H), CMT64(M) and CMT170(L) cell lines, selected from a spontaneous mouse lung adenocarcinoma, with high-, middle- or low-metastatic potential have been characterized in vivo. In this study, the comprehensive protein expression profiles of the CMT cell lines were analyzed at passages 5, 15 and 35 in order to assess the cell line stability. During the passages 5 to 15, the expression profiles of CMT cells remained reasonably stable as evidenced by only 0.7%, 3.9% and 1.1% proteins changed in CMT167(H), CMT64(M) and CMT170(L) respectively. However, the number of differentially expressed proteins were considerably increased at passage 35 in CMT64(M) and CMT170(L) while CMT167(H) remained stable. Based on our selection criteria, 22, 109 and 84 spots in CMT167(H), CMT64(M) and CMT170(L) were selected for protein identification by MS and 99 unique proteins were identified. Bioinformatics analysis indicated that most of these proteins participate in cellular metabolism. In conclusion, proteomics was found to be a useful tool for assessing differences in cell line stability. This approach provided a tool to select the best cell line and optimal subculture period for studies of cancer related phenomena and for testing the effect of potential anticancer drugs.

Leishmania is a parasitic protozoan of trypanosomatidae family which causes a wide spectrum of diseases ranging from self-healing cutaneous lesions to deadly visceral forms. In endemic areas, field trials of different preparations of Leishmania total antigen were tested as leishmaniasis vaccine. Two preparations of killed Leishmania major were produced In Iran, which were heat-killed vaccine called autoclaved L. major (ALM) and thimerosal-treated freeze-thawed vaccine called killed L. major (KLM). In this study, the protein content of both ALM and KLM were compared with that of freshly harvested intact L. major promastigotes using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

The main principles of higher-order protein oligomerization are elucidated by many structural and biophysical studies. An astonishing number of proteins self-associate to form dimers or higher-order quaternary structures which further interact with other biomolecules to elicit complex cellular responses. In this study, we describe a simple and convenient approach to determine the oligomeric state of purified protein complexes that combines implementation of a novel form of clear-native gel electrophoresis and size exclusion chromatography in line with multi-angle light scattering. Here, we demonstrate the accuracy of this ensemble approach by characterizing the previously established pentameric state of the intracellular domain of serotonin type 3A (5-HT3A) receptors.

Obstructive sleep apnea (OSA) affects an estimated 20% of adults worldwide and has been associated with electrical and structural abnormalities of the atria, although the molecular mechanisms are not well understood. Here, we used two-dimensional polyacrylamide gel electrophoresis (2D PAGE) coupled with nanoliquid chromatography-tandem mass spectrometry (nanoLC-MS/MS) to investigate the proteins that are dysregulated in the atria from severe and moderate apnea when compared to control. We found enzymes involved in the glycolysis, beta-oxidation, electron transport chain and Krebs cycle to be down-regulated. The data suggested that the dysregulated proteins may play a role in atrial pathology developing via chronic obstructive apnea and hypoxia. Our results are consistent with our previous 1D-PAGE and nanoLC-MS/MS study (Channaveerappa et al, J Cell Mol Med. 2017), where we found that some aerobic and anaerobic glycolytic and Krebs cycle enzymes were down-regulated, suggesting that apnea may be a result of paucity of oxygen and production of ATP and reducing equivalents (NADH). The 2D-PAGE study not only complements our current study, but also advances our understanding of the OSA. The complete mass spectrometry data are available via ProteomeXchange with identifier PXD011181.

Muscovy duck reovirus (MDRV) belongs to the Orthoreovirus genus of the Reoviridae family, which is a significant poultry pathogen leading to high morbidity and mortality in ducklings. However, the pathogenesis of the virus is not well understood. In the present study, two-dimensional (2D) polyacrylamide gel electrophoresis (PAGE) combined with LC-MS-MS was used to identify differentially expressed proteins between Muscovy duck embryo fibroblasts (MDEF) infected with virulent (MV9710 strain) and attenuated (CA strain) MDRV and non-infected MDEFs. A total of 115 abundant protein spots were identified. Of these, 59 of differentially expressed proteins were detected, with functions in metabolism and utilization of carbohydrates and nucleotides, anti-stress, and regulation of immune and cellular process. GO analysis of the identified proteins showed that they belonged to the classes molecular function (141 proteins), cellular component (62 proteins), and biological process (146 proteins). The results were validated by qRT-PCR, which suggests that the analysis method of 2D PAGE combined with LC-MS-MS used in this study is reliable. This study lays a foundation for further investigation of the biology of MDRV infection in MDEF.

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.

Tuberculosis is an infectious bacterial disease in humans caused primarily by Mycobacterium tuberculosis, and infects one-third of the world's total population. Mycobacterium bovis bacillus Calmette-Guérin (BCG) vaccine has been widely used to prevent tuberculosis worldwide since 1921. Membrane proteins play important roles in various cellular processes, and the protein-protein interactions involved in these processes may provide further information about molecular organization and cellular pathways. However, membrane proteins are notoriously under-represented by traditional two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and little is known about mycobacterial membrane and membrane-associated protein complexes. Here we investigated M. bovis BCG by an alternative proteomic strategy coupling blue native PAGE to liquid chromatography tandem mass spectrometry (LC-MS/MS) to characterize potential protein-protein interactions in membrane fractions.

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is a widely utilized technique for macromolecule and protein analysis. While multiple methods exist to visualize the separated protein bands on gels, one of most popular methods of staining the proteins is with Coomassie dye. A more recent approach is to use Bio-Rad stain-free technology for visualizing protein bands with UV light and achieve similar or greater sensitivity than that of Coomassie dye. Here, we developed a method to further amplify the sensitivity of stain-free gels using carboxyfluorescein succinimidyl ester (CFSE) staining. We compared our novel method using foetal bovine serum samples with Coomassie dye, standard stain-free gels, and silver staining. Our results show that while silver staining remains a gold-standard method in terms of sensitivity; CFSE staining of samples prior to use with stain-free gels results in a 10-100-fold increase in sensitivity over Coomassie staining and the standard stain-free method. Our method offers a sensitivity similar to that of silver staining which is compatible with downstream mass spectrometry, and therefore more advantageous for further retrieval and analysis of macromolecules in bands.

The social amoeba Dictyostelium discoideum was instrumental in the discovery and early characterization of inositol pyrophosphates, a class of molecules possessing highly-energetic pyrophosphate bonds. Inositol pyrophosphates regulate diverse biological processes and are attracting attention due to their ability to control energy metabolism and insulin signalling. However, inositol pyrophosphate research has been hampered by the lack of simple experimental procedures to study them. The recent development of polyacrylamide gel electrophoresis (PAGE) and simple staining to resolve and detect inositol pyrophosphate species has opened new investigative possibilities. This technology is now commonly applied to study in vitro enzymatic reactions. Here we employ PAGE technology to characterize the D. discoideum inositol pyrophosphate metabolism. Surprisingly, only three major bands are detectable after resolving acidic extract on PAGE. We have demonstrated that these three bands correspond to inositol hexakisphosphate (IP₆ or Phytic acid) and its derivative inositol pyrophosphates, IP₇ and IP₈. Biochemical analyses and genetic evidence were used to establish the genuine inositol phosphate nature of these bands. We also identified IP₉ in D. discoideum cells, a molecule so far detected only from in vitro biochemical reactions. Furthermore, we discovered that this amoeba possesses three different inositol pentakisphosphates (IP₅) isomers, which are largely metabolised to inositol pyrophosphates. Comparison of PAGE with traditional Sax-HPLC revealed an underestimation of the cellular abundance of inositol pyrophosphates by traditional methods. In fact our study revealed much higher levels of inositol pyrophosphates in D. discoideum in the vegetative state than previously detected. A three-fold increase in IP₈ was observed during development of D. discoideum a value lower that previously reported. Analysis of inositol pyrophosphate metabolism using ip6k null amoeba revealed the absence of developmentally-induced synthesis of inositol pyrophosphates, suggesting that the alternative class of enzyme responsible for pyrophosphate synthesis, PP-IP₅K, doesn't' play a major role in the IP₈ developmental increase.

Arabidopsis (Arabidopsis thaliana) proteins were isolated from five tissues (leaf, stem, root, seed and callus), and separated by two-dimensional gel electrophoresis (2-DE). 2-DE was carried out by immobilized pH gradient (IPG) in the first dimension, and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the second dimension. With the aid of comigrated five-marker proteins, the patterns of 2-DE gels for each tissue were graphically combined by a computer into a single synthetic image for the integrated Arabidopsis protein spots. The protein spot images, altogether 4763, were characterized by both molecular mass and isoelectric point. Partial amino(N)-terminal sequences of 101 protein spots were analyzed by Edman degradation. Fifty seven proteins were partially sequenced and 46 proteins appeared to have blocked N-termini. Deblocking by hydrazine vapor was carried out on 14 proteins and two of them were found to be pyroglutamyl-blocked N-termini. Forty seven new proteins were found by the present investigation.

We developed a novel, hybrid method combining both blue-native (BN-PAGE) and clear-native (CN-PAGE) polyacrylamide gel electrophoresis, termed BCN-PAGE, to perform in-gel activity stains on the mitochondrial electron transport chain (ETC) complexes in skin fibroblasts.

The intention of this review is to provide an overview of current methodologies employed in the rapidly developing field of ocular proteomics with emphasis on sample preparation, two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and mass spectrometry (MS). Appropriate sample preparation for the diverse range of cells and tissues of the eye is essential to ensure reliable results. Current methods of protein staining for 2D-PAGE, protein labelling for two-dimensional difference gel electrophoresis, gel-based expression analysis and protein identification by MS are summarised. The uses of gel-free MS-based strategies (MuDPIT, iTRAQ, ICAT and SILAC) are also discussed. Proteomic technologies promise to shed new light onto ocular disease processes that could lead to the discovery of strong novel biomarkers and therapeutic targets useful in many ophthalmic conditions.

Visceral leishmaniasis is a parasitic infection caused by Lesihmania donovani complex and transmitted by the bite of the phlebotomine sand fly. It is an endemic disease in many developing countries with more than 90% of the cases occurring in Bangladesh, India, Nepal, Sudan, Ethiopia and Brazil. The disease is fatal if untreated. The disease is conventionally diagnosed by demonstrating the intracellular parasite in bone marrow or splenic aspirates. This study was carried out to discover differentially expressed proteins which could be potential biomarkers.

Two-dimensional gel-electrophoresis (2-DE) images show the expression levels of several hundreds of proteins where each protein is represented as a blob-shaped spot of grey level values. The spot detection, that is, the segmentation process has to be efficient as it is the first step in the gel processing. Such extraction of information is a very complex task. In this paper, we propose a novel spot detector that is basically a morphology-based method with the use of a seeded region growing as a central paradigm and which relies on the spot correlation information. The method is tested on our synthetic as well as on real gels with human samples from SWISS-2DPAGE (two-dimensional polyacrylamide gel electrophoresis) database. A comparison of results is done with a method called pixel value collection (PVC). Since our algorithm efficiently uses local spot information, segments the spot by collecting pixel values and its affinity with PVC, we named it local pixel value collection (LPVC). The results show that LPVC achieves similar segmentation results as PVC, but is much faster than PVC.

Due to the constant search for reliable methods to investigate glycoproteins in complex biological samples, an alternative approach combining affinity enrichment with rapid and sensitive analysis on-a-chip is presented. Glycoproteins were specifically captured by lectin-coated magnetic beads, eluted by competitive sugars, and investigated with microchip capillary gel electrophoresis (MCGE), i.e., CGE-on-a-chip. We compared our results to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) data, which turned out to be in very good agreement. While SDS-PAGE offers the possibility of subsequent mass spectrometric analysis of captured and separated analytes, MCGE scores with time savings, higher throughput, and lower sample consumption as well as quality control (QC) and process analytical technology (PAT) applicability. Due to these advantages, a lectin-based glycoprotein capture protocol can easily be optimized. In our case, two different types of magnetic beads were tested and compared regarding lectin binding. The selectivity of our strategy was demonstrated with a set of model glycoproteins, as well as with human serum and serum depleted from high-abundance proteins. The specificity of the capturing method was investigated revealing to a certain degree an unspecific binding between each sample and the beads themselves, which has to be considered for any specific enrichment and data interpretation. In addition, two glycoproteins from Trichoderma atroviride, a fungus with mycoparasitic activity and only barely studied glycoproteome, were enriched by means of a lectin and so identified for the first time. Graphical abstract Glycoproteins from biological samples were detected by microchip capillary gel electrophoresis after lectin affinity enrichment using magnetic beads and elution with respective competitive monosaccharides.