A novel 2D-electrophoresis method for the simultaneous visualization of phosphorylated and O-GlcNAcylated proteoforms of a protein.

Post-translational modifications (PTMs), such as phosphorylation and O-N-acetyl-ß-d-glucosaminylation (O-GlcNAcylation), are involved in the fine spatiotemporal regulation of protein functions, and their dynamic interplay is at the heart of protein language. The coexistence of phosphorylation and O-GlcNAcylation on a protein leads to the diversification of proteoforms. It is therefore essential to decipher the phosphorylation/O-GlcNAcylation interplay on protein species that orchestrates cellular processes in a specific physiological or pathophysiological context. However, simultaneous visualization of phosphorylation and O-GlcNAcylation patterns on a protein of interest remains a challenge. To map the proteoforms of a protein, we have developed an easy-to-use two-dimensional electrophoresis method with a single sample processing permitting simultaneous visualization of the phosphorylated and the O-GlcNAcylated forms of the protein of interest. This method, we termed 2D-WGA-Phos-tag-PAGE relies on proteoforms retardation by affinity gel electrophoresis. With this novel approach, we established the cartography of phospho- and glycoforms of αB-crystallin and desmin in the whole extract and the cytoskeleton protein subfraction in skeletal muscle cells. Interestingly, we have shown that the pattern of phosphorylation and O-GlcNAcylation depends of the subcellular subfraction. Moreover, we have also shown that proteotoxic stress condition increased the complexity of the pattern of PTMs on αB-crystallin.

Early hepatic proteomic signatures reveal metabolic changes in high-fat-induced obesity in rats.

The prevalence of diet-related obesity is increasing dramatically worldwide, making it important to understand the associated metabolic alterations in the liver. It is well known that obesity is a multifactorial condition that is the result of complex integration between many gene expressions and dietary factors. Obesity alone or in conjunction with other chronic diseases such as diabetes and insulin resistance causes many health problems and is considered a major risk factor for developing non-alcoholic steatohepatitis (NASH) and cirrhosis. In this study, we aimed to understand the molecular mechanisms underlying early hepatic changes in the pathophysiology of high-fat diet (HFD)-induced abdominal obesity in rats. Hepatic protein profiles of normal diet and HFD-induced obesity for 24 weeks were analysed using two-dimensional differential gel electrophoresis (DIGE) and protein identification by MS. Fifty-two proteins were identified by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF), and computer-assisted DIGE image software analysis showed that eighteen major proteins were significantly differentially expressed between comparable groups, with 2·0­4·0-fold change/more (P < 0·01). These proteins are regulated in response to a HFD, and differentially expressed proteins are involved in key metabolic pathways such as lipid metabolism, energy metabolism, detoxification, urea cycle and hepatic Ca homoeostasis. In addition, Western blot and immunohistochemistry of liver-specific arginase-1 (Arg-1) showed significant increased expression in the liver of high-fat-fed rats (P < 0·01). Further, Arg-1 expression was correlated with NASH patients with obesity-related fibrosis (F0­F4). It is concluded that high-fat content may affect changes in liver pathways and may be a therapeutic target for obesity-related liver disease. Arg-1 expressions may be a potential pathological marker for assessing the progression of the disease.

Recognition of immune reactive proteins as a potential multiepitope vaccine candidate of Taenia solium cysticerci through proteomic approach.

Metacestode, the larva of Taenia solium, is the causative agent for neurocysticercosis (NCC), which causes epilepsy. The unavailability of a vaccine against human NCC is a major cause for its widespread prevalence across the globe. Therefore, the development of a reliable vaccine against NCC is the need of the hour. Employing a combination of proteomics and immunoinformatics, we endeavored to formulate a vaccine candidate. The immune reactive cyst fluid antigens of T. solium were identified by immune-blotting two-dimensional gels with NCC patient's sera, followed by Matrix-assisted laser desorption-ionization analysis. We performed a detailed proteomic study of these immune reactive proteins by utilizing immune-informatics tools, identified the nontoxic, nonallergic, B-cell epitopes, and collected epitopes with the least sequence homology with human and other Taenia species. These epitopes were joined through linkers to construct a multiepitope vaccine. Different physiochemical parameters such as molecular weight (23.82 kDa), instability (39.91), and aliphatic index (49.61) were calculated to ensure the stability of the linked peptides vaccine. The vaccine demonstrated stable interactions with different immune receptors like Toll-like receptor 4 and IgG confirming that it will effectively stimulate the host immune response. We anticipate that our designed B-cell linear epitope-based vaccine will show promising results in in vitro and in vivo assays. This study provides a platform that would be useful to develop other suitable vaccine candidates to prevent helminthic neglected tropical diseases in near future.

Analysis and comparison of tear protein profiles in dogs using different tear collection methods.

BACKGROUND: Tear proteomic analysis has become an important tool in medical and veterinary research. The tear collection method could influence the tear protein profile. This study aims to evaluate the protein profiles of dog tears collected using microcapillary tubes (MT), Schirmer tear strips (ST), and ophthalmic sponges (OS). METHODS: The tear samples were collected using MT, ST, and OS. Tear protein profiles were analyzed using two-dimensional electrophoresis (2-DE) and the different protein spots' expression was compared. Fourteen protein spots were identified using liquid chromatography-tandem mass spectrometry (LC-MS/MS). RESULTS: Tear protein concentrations ranged from 2.80 to 4.03 µg/µL, with no statistically significant differences among collection methods. Protein expression in each collection method differed in terms of both the number and intensity of the spots. There were 249, 327, and 330 protein spots found from tears collected with MT, ST, and OS, respectively. The proteins albumin, haptoglobin, and lactoferrin identified from OS were found to have higher spot intensities than other methods of collection. The use of MT demonstrated the downregulation of nine proteins. CONCLUSIONS: The recent study supported that tear protein analysis is affected by different tear collection methods. Although ST is commonly used for tear collection, it provides insufficient information to study particular tear proteins.

Prenatal diagnosis of mucopolysaccharidoses type II by two-dimensional electrophoresis and mass spectrometry in amniotic fluid.

AIM: To introduce a quantitative determination of heparan sulfate and dermatan sulfate by mass spectrometry and to compare it with two-dimensional electrophoresis of the glycosaminoglycans in the amniotic fluid for the prenatal diagnosis of mucopolysaccharidoses type II (MPS II). METHODS: Thirty pregnancies each with single fetus were subjected to amniocentesis at 16 weeks: 10 with a previously affected MPS II infant and 20 as controls. Prenatal diagnosis was done by both mass spectrometry two two-dimensional electrophoresis. RESULTS: Two-dimensional electrophoresis showed four affected with MPS II and six unaffected fetuses. Mass spectrometry verified these results. CONCLUSION: Two-dimensional electrophoresis of the glycosaminoglycans in amniotic fluid is a good qualitative method and mass spectrometry is a new accurate quantitative method for prenatal diagnosis of MPS II. Quantitative determination of glycosaminoglycans in amniotic fluid by mass spectrometry is both rapid and accurate. Prenatal diagnosis is recommended for at risk pregnancies and mass spectrometry offers speed and quantitation.

Multielemental, Nutritional, and Proteomic Characterization of Different Lupinus spp. Genotypes: A Source of Nutrients for Dietary Use.

Among grain pulses, lupins have recently gained considerable interest for a number of attractive nutritional attributes relating to their high protein and dietary fiber and negligible starch contents. The seeds of Lupinus albus (cv. Multitalia and Luxor, and the Modica ecotype); L. luteus (cv. Dukat, Mister, and Taper); and L. angustifolius (cv. Sonet) analyzed in this study were deposited within the germplasm collection of the Research Centre for Cereal and Industrial Crops of Acireale and were sowed in East Sicily in 2013/14. The collected seeds were analyzed for their multielemental micro- and macronutrient profiles, resulting in a wide variability between genotypes. Lupin seed flour samples were subjected to a defatting process using supercritical CO2, with oil yields dependent on the species and genotype. We determined the fatty acid profile and tocopherol content of the lupin oil samples, finding that the total saturated fatty acid quantities of different samples were very close, and the total tocopherol content was about 1500.00 µg/g FW. The proteomic analysis of the defatted lupin seed flours showed substantial equivalence between the cultivars of the same species of Lupinus albus and L. luteus. Moreover, the L. angustifolius proteome map showed the presence of additional spots in comparison to L. albus, corresponding to α-conglutins. Lupin, in addition to being a good source of mineral elements, also contributes vitamin E and, thanks to the very high content of gamma-tocopherols, demonstrates powerful antioxidant activity.

Differential mitochondrial proteomic analysis of A549 cells infected with avian influenza virus subtypes H5 and H9.

BACKGROUND: Both the highly pathogenic avian influenza (HPAI) H5N1 and low pathogenic avian influenza (LPAI) H9N2 viruses have been reported to cross species barriers to infect humans. H5N1 viruses can cause severe damage and are associated with a high mortality rate, but H9N2 viruses do not cause such outcomes. Our purpose was to use proteomics technology to study the differential expression of mitochondrial-related proteins related to H5N1 and H9N2 virus infections. METHODS: According to the determined viral infection titer, A549 cells were infected with 1 multiplicity of infection virus, and the mitochondria were extracted after 24 h of incubation. The protein from lysed mitochondria was analyzed by the BCA method to determine the protein concentration, as well as SDS-PAGE (preliminary analysis), two-dimensional gel electrophoresis, and mass spectrometry. Differential protein spots were selected, and Western blotting was performed to verify the proteomics results. The identified proteins were subjected to GO analysis for subcellular localization, KEGG analysis for functional classification and signaling pathways assessment, and STRING analysis for functional protein association network construction. RESULTS: In the 2-D gel electrophoresis analysis, 227 protein spots were detected in the H5N1-infected group, and 169 protein spots were detected in the H9N2-infected group. Protein spots were further subjected to mass spectrometry identification and removal of redundancy, and 32 differentially expressed proteins were identified. Compared with the H9N2 group, the H5N1-infected group had 16 upregulated mitochondrial proteins and 16 downregulated proteins. The differential expression of 70-kDa heat shock protein analogs, short-chain enoyl-CoA hydratase, malate dehydrogenase, and ATP synthase was verified by Western blot, and the results were consistent with the proteomics findings. Functional analysis indicated that these differentially expressed proteins were primarily involved in apoptosis and metabolism. CONCLUSIONS: Compared with their expression in the H9N2 group, the differential expression of eight mitochondrial proteins in the H5N1 group led to host T cell activation, antigen presentation, stress response, ATP synthesis and cell apoptosis reduction, leading to higher pathogenicity of H5N1 than H9N2.

Proteomic analysis reveals that naturally produced citral can significantly disturb physiological and metabolic processes in the rice blast fungus Magnaporthe oryzae.

Rice blast (Magnaporthe oryzae), a major fungal disease in rice producing areas all over the world as well as in China, seriously affects the safety of rice production. Citral, a mixture of Z/E and trans isomers, is a natural acycloid monoterpene compound with good bacteriostatic effect on rice blast. To further investigate the underlying molecular mechanism, a comparative proteomics analysis was conducted between citral-treated and non-treated M. oryzae spores through two-dimensional gel electrophoresis and MALDI-TOF mass spectrometry. Our analysis identified 1600-1800 proteins from M. oryzae ZB15, of which 147 were differentially expressed in 100 µg/mL citral-treated samples relative to the control group. Among these differentially expressed proteins (DEPs), 40 proteins showed significantly different expression. GO enrichment and NCBI conserved domains database analysis showed that the main groups of the cellular component were cytoplasm (23.33%), and the major molecular function categories were ion binding (31.37%), and the major categories of biological processes included small molecule metabolic process (22.22%) and transport (13.89%). Further analysis found that down-regulated proteins included the tubulin α chain, ATP synthase subunit ß and malate dehydrogenase, while the tubulin ß, enolase were upregulated. These DEPs could possibly limit the availability of energy required for many cellular processes and result in various physiological adaptions of M. oryzae. This study represents the first proteomic analysis of M. oryzae treated by citral and will help to uncover the mode-of-action of this biologically active compound against M. oryzae. These findings have practical implications with respect to the use of citral for fungal disease control.

2D electrophoresis image brightness correction based on gradient interval histogram.

BACKGROUND: Two-dimensional electrophoresis (2DE) is one of the most widely applied techniques in comparative proteomics. The basic task of 2DE is to identify differential protein expression by quantitative analysis of 2DE images. To reduce the errors of spot quantification in 2DE images, a novel brightness correction method based on gradient interval histogram (GIH) is proposed in this paper. RESULTS: First, GIH equalization is proposed to enhance the protein spot edges, especially the weak protein spots in the 2DE image. Second, to eliminate the overall brightness shift, GIH matching is applied to the 2DE images that need to be compared. Finally, the proposed method is verified by subjective quality evaluation and quantitative analysis of protein spots in real 2DE images. CONCLUSIONS: The experimental results show that the average error of the quantification of corresponding protein spots in the resulting image pairs is less than 3%, which is significantly superior to that of the existing methods.

Differential expression of MAP3K7 and TROPONIN C proteins and related perturbations in renal amyloidosis.

OBJECTIVES: Renal amyloidosis (RA) is a rare protein misfolding disorder that prompts progressive renal insufficiency. This study aimed to decipher proteomic changes in human sera to understand the pathophysiology and molecular mechanisms underlying the disease development, hence assisting in the diagnosis of RA. METHODS: Serum proteomic analysis was performed using a gel-based approach followed by MALDI-TOF MS. RA patients with age and sex matched healthy volunteers were recruited from Max Super Speciality Hospital, New Delhi, India. RESULTS: Proteome profiles of serum revealed eight differentially expressed proteins namely, Zinc finger protein 624, Protein FAM183A, Calcium-binding mitochondrial carrier protein Scamc-3, V-type proton ATPase 116 kDa subunit A isoforms 2, Protein TXNRD3NB, ATP - dependent RNA helicase, Troponin C and Mitogen-activated protein kinase kinase kinase 7. These proteins were reported first time in RA. The increased levels of MAP3K7 and TROPONIN C were validated by bio-layer interferometry and their diagnostic accuracy was evaluated by ROC curve analysis. The differentially expressed proteins were predominantly associated with vesicular trafficking, transcriptional regulation, metabolic processes, apoptotic process and mitochondrial metabolism. CONCLUSION: The results indicate that these proteomic signatures may be considered as potential molecular targets for RA diagnostics and therapeutics subject to validation on large sample size. Abbreviations: AßP= Amyloid-beta protein, Aß=Amyloid-beta, AL= Light chain amyloidosis, AA= Amyloid A, ALECT2= LECT2 amyloidosis, APS= Ammonium persulfate CKD= Chronic Kidney Diseases, EBRT= external beam radiation therapy, ESRD= End-Stage Kidney Disease, Glis2= Gli-similar 2, JNK= c-Jun NH 2-terminal kinase, MAPK= Mitogen-Activated Protein Kinase, MM=Multiple Myeloma, PHD= Prolyl hydroxylase, RA = Renal Amyloidosis, SAA= Serum Amyloid A, SD= Standard Deviation, Sepp= Selenoprotein, SCC= Squamous cell carcinoma, SDS= Sodium dodecyl sulfate, TEMED = tetramethyl ethylenediamine, TGF-Beta-1=Transforming growth factor- Beta-1, Trx = Thioredoxin, TrxR= Thioredoxin reductase.

Reduced expression of annexin A1 promotes gemcitabine and 5-fluorouracil drug resistance of human pancreatic cancer.

Intrinsic chemoresistance is the main reason for the failure of human pancreatic ductal adenocarcinoma (PDAC) therapy. To identify the candidate protein, we compared the protein expression profiling of PDAC cells and its distinct surviving cells following primary treatment with gemcitabine (GEM) and 5-fluorouracil (5-FU) by two-dimensional electrophoresis combined with liquid chromatography-mass spectrometry or mass spectrometry. A total of 20 differentially expressed proteins were identified, and annexin A1 (ANXA1) was analyzed for further validation. The functional validation showed that the downregulation of ANXA1 contributes to GEM and 5-FU resistance in PDAC cells through protein kinase C/c-Jun N-terminal kinase/P-glycoprotein signaling pathway. Our findings provide a platform for the further elucidation of the underlying mechanisms of PDAC intrinsic chemoresistance and demonstrated that ANXA1 may be a valid marker for anticancer drug development.

Differentially expressed proteins in the intestine of Cynoglossus semilaevis Günther following a Shewanella algae challenge.

Fish intestine is an important constituent of the mucosal immune system. The gut and gut-associated lymphoid tissue construct a local immune environment. A Shewanella algae strain was previously reported to be a pathogen causing ascitic disease accompanied with intestinal inflammation in Cynoglossus semilaevis. This study aimed to investigate the intestine immune response in C. semilaevis to S. algae infection at the protein level. Two-dimensional electrophoresis coupled with mass spectrometry proteomics was utilized to compare protein expression in the intestines from normal and S. algae-infected C. semilaevis. A total of 70 differentially expressed proteins (DEPs), consisting of 16 upregulated and 54 downregulated proteins, were identified in the intestine tissue of C. Semilaevis. These protein expression changes were further validated using western blot analysis and quantitative real-time PCR. Gene ontology enrichment analysis showed that these 70 DEPs could be assigned across three categories: "cellular components", "molecular function", and "biological process". Forty-one DEPs (six up-regulated and 35 down-regulated proteins) related to metabolic processes were identified. In addition, 20 DEPs (eight up-regulated and 12 down-regulated proteins) related to stress and immune responses were identified. A protein-protein interaction network generated by the STRING (Search Tool for the Retrieval of Interacting Genes/protein) revealed that 30 DEPs interacted with one another to form an integrated network. Among them, 29 DEPs were related to stress, immune, and metabolism processes. In the network, some of the immune related proteins (C9, FGB, KNG1, apolipoprotein A-IV-like, and PDIA3) were up-regulated and most DEPs involved in metabolism processes were down-regulated. These results indicate that the immune defense response of the intestine was activated and the intestinal function associated with metabolism processes was disturbed. This study provides valuable information for further research into the functions of these DEPs in fish.

The intestinal milieu influences the immunoproteome of male and female Heligmosomoides polygyrus bakeri L4 stage.

The gastrointestinal nematode Heligmosomoides polygyrus bakeri shows enhanced survival in mice with colitis. As the antibody response plays an important role in antiparasitic immunity, antibodies against male and female L4 H. polygyrus were examined in mice with and without colitis. Levels of specific antibodies in the mucosa and serum were determined by enzyme-linked immunosorbent assay and immunogenic proteins of male and female parasites were identified using 2D electrophoresis and mass spectrometry. The function of identified proteins was explored with Blast2Go. Nematodes in mice with colitis induced higher levels of specific immunoglobulin G (IgG1) and IgA, a lower level of IgE in the small intestine and a higher level of IgE in serum against female L4. Infected mice with colitis recognized 12 proteins in male L4 and 10 in female L4. Most of the recognized proteins from male L4 were intermediate filament proteins, whereas the proteins from female L4 were primarily actins and galectins. Nematodes from mice with colitis were immunogenically different from nematodes from control mice. This phenomenon gives new insights into helminth therapy as well as host-parasite interactions.

Proteomic analysis of the inhibitory effect of the butanolic fraction of Jacquinia macrocarpa on Fusarium verticillioides.

Jacquinia macrocarpa, a plant native to northwestern Mexico, has an inhibitory effect against phytopathogenic fungi. Previous studies have shown that the butanolic extract of J. macrocarpa causes retardation and atrophy in mycelial growth of Fusarium verticillioides. However, the action mechanism of this extract is unknown. We used a proteomics approach to understand the inhibitory effect of J. macrocarpa butanolic extract, based on differential protein accumulation in F. verticillioides. Proteins were extracted from F. verticillioides cultured in Czapek broth with and without 202.12 µg/mL (IC50) of butanolic extract of J. macrocarpa. Thirty-eight protein spots showing statistically significant changes (ANOVA, p < 0.01) and at least a 2-fold change in abundance between experimental conditions were analyzed by mass spectrometry. Identified proteins were grouped into different biological processes according to Gene Ontology, among them were amino acid metabolism, protein folding and stabilization, protein degradation, protein transport, carbohydrate metabolism, oxidative stress response, and miscellaneous. This work is the first report of changes in the proteomic profile of F. verticillioides exposed to the J. macrocarpa extract. This information provides new insights into the inhibitory mechanism of the extract and represents a starting point for dissection of the fungal response against the J. macrocarpa extract components.

Agaricus bisporus Crude Extract: Characterization and Analytical Application.

In the present work crude Agaricus bisporus extract (ABE) has been prepared and characterized by its tyrosinase activity, protein composition and substrate specificity. The presence of mushroom tyrosinase (PPO3) in ABE has been confirmed using two-dimensional electrophoresis, followed by MALDI TOF/TOF MS-based analysis. GH27 alpha-glucosidases, GH47 alpha-mannosidases, GH20 hexosaminidases, and alkaline phosphatases have been also detected in ABE. ABE substrate specificity has been studied using 19 phenolic compounds: polyphenols (catechol, gallic, caffeic, chlorogenic, and ferulic acids, quercetin, rutin, dihydroquercetin, l-dihydroxyphenylalanine, resorcinol, propyl gallate) and monophenols (l-tyrosine, phenol, p-nitrophenol, o-nitrophenol, guaiacol, o-cresol, m-cresol, p-cresol). The comparison of ABE substrate specificity and affinity to the corresponding parameters of purified A. bisporus tyrosinase has revealed no major differences. The conditions for spectrophotometric determination have been chosen and the analytical procedures for determination of 1.4 × 10-4-1.0 × 10-3 M l-tyrosine, 3.1 × 10-6-1.0 × 10-4 M phenol, 5.4 × 10-5-1.0 × 10-3 M catechol, 8.5 × 10-5-1.0 × 10-3 M caffeic acid, 1.5 × 10-4-7.5 × 10-4 M chlorogenic acid, 6.8 × 10-5-1.0 × 10-3 M l-DOPA have been proposed. The procedures have been applied for the determination of l-tyrosine in food supplements, l-DOPA in synthetic serum, and phenol in waste water from the food manufacturing plant. Thus, we have demonstrated the possibility of using ABE as a substitute for tyrosinase in such analytical applications, as food supplements, medical and environmental analysis.

Improved two-dimensional electrophoretic mapping of Japanese human hair proteins; application to curved and straight Japanese human hairs; and protein identification by MALDI MS and MS/MS quadrupole time-of-flight mass spectrometry.

OBJECTIVES: To evaluate improved protein extraction and two-dimensional electrophoresis (2DE) separation methods with Japanese reference human hair (JRH); to determine whether fibre curvature is related to protein composition in curly and straight Japanese women's human hair (JHH) samples; and to identify proteins from JRH 2DE maps and expression differences between curly and straight JHH. METHODS: Hair keratin and keratin-associated proteins (KAPs) were extracted intact with dithiothreitol or tris(2-carboxyethyl) phosphine from JRH or from curved or straight JHH. Extracted proteins were isoelectric-focused on first-dimensional pH gradient gel strips, then separated by molecular weight on laboratory-made, second-dimension, large format gels. The software compared protein abundance between duplicate 2DE gels of curved and straight JHH. Thirty-eight proteins from a JRH 2DE gel were enzyme-cleaved for MALDI-TOF-MS analysis to determine peptide composition, and where possible, de novo sequencing gave peptide sequence data. An in-house human hair protein database incorporating ninety-eight annotated protein sequences assisted MS analysis. RESULTS: 2DE gels of tris(2-carboxyethyl) phosphine-extracted JRH improved keratin and KAP resolution and number compared to those of dithiothreitol-extracted JRH and published commercially made second-dimensional gels. Silver-stained 2DE gels of the straight or curved JHH sets were remarkably similar. Over-staining to reveal basic proteins caused poor resolution of the major acidic protein classes. Software comparisons of fifty-nine resolved proteins revealed two were significantly different in abundance between curved and straight hairs but in insufficient amounts for MS analysis. MS identified twelve proteins from a JRH CBBG-stained 2DE gel: six type II keratins, three type I keratins and three high sulphur proteins. A further eight were potential conformational isoforms and isoelectric variants of the identified proteins bringing the total to twenty identified or partially identified proteins. CONCLUSION: Root-end human hair extraction with tris(2-carboxyethyl) phosphine improves protein resolution and visualizes more proteins on large format 2DE gels. The two minor protein differences between duplicate straight or curved JHH 2DE gels were unlikely to change fibre structure from straight to curved hair. MS results confirmed that multiple isoforms exist of various hair proteins. Low sequence coverage prevented distinction between members in rows of homologous protein spots of similar molecular weight.

OBJECTIFS: évaluer l'amélioration de l'extraction de protéines et les méthodes de séparation bidimensionnelle par électrophorèse (2DE) avec des cheveux humains de référence Japonais (JRH), déterminer si la courbure de la fibre est liée à la composition protéique dans les échantillons de cheveux humains des Japonaises (JHH) bouclés et raides et identifier les protéines issues des cartes JRH 2DE et les différences d'expression entre les JHH bouclés et raides. MÉTHODES: la kératine des cheveux et les protéines associées à la kératine (KAP) ont été extraites intactes avec du dithiothréitol ou du tris (2-carboxyéthyl) phosphine des JRH ou des JHH bouclés ou raides. Les protéines extraites ont subi une focalisation isoélectrique sur des bandes de gel à gradient de pH unidimensionnelles, puis ont été séparées par poids moléculaire sur des gels bidimensionnels de grand format, fabriqués en laboratoire. Le logiciel a comparé l'abondance des protéines entre les deux duplicatas de gels 2DE des JHH bouclés et raides. Trente-huit protéines provenant d'un gel 2DE JRH ont été clivés par enzyme pour l'analyse MALDI-TOF-MS afin de déterminer la composition des peptides, et dans la mesure du possible, un séquençage de novo a donné des données de séquence des peptides. Une base de données interne des protéines capillaires humaines incorporant 98 séquences de protéines annotées a aidé l'analyse MS. RÉSULTATS: les gels 2DE de JRH extraits par le tris (2-carboxyéthyl) ont amélioré la résolution et le nombre de la kératine et du KAP par rapport à ceux du JRH extrait par le dithiothréitol et des gels bidimensionnels fabriqués commercialement. Les gels 2DE à coloration argentée des ensembles de JHH raides ou bouclés étaient remarquablement similaires. La sur-coloration pour révéler les protéines de base a provoqué une mauvaise résolution des principales classes de protéines acides. Les comparaisons logicielles des 59 protéines résolues ont révélé que deux présentaient une différence significative d'abondance entre les cheveux bouclés et raides, mais en quantités insuffisantes pour une analyse MS. La MS a identifié douze protéines provenant d'un gel 2DE coloré CBBG JRH : six kératines de type II, trois kératines de type I et trois protéines à forte teneur en soufre. Huit autres étaient des isoformes conformationnels potentiels et des variantes isoélectriques des protéines identifiées, ramenant le total à 20 protéines identifiées ou partiellement identifiées. CONCLUSION: l'extraction des cheveux humains à la racine avec du tris (2-carboxyéthyl) phosphine améliore la résolution des protéines et permet de visualiser plus de protéines sur les gels 2DE grand format. Les deux différences de protéines mineures entre les duplicatas des gels 2DE JHH raides ou bouclés étaient peu susceptibles de changer la structure des fibres de cheveux raides à bouclés. Les résultats de la MS ont confirmé qu'il existe plusieurs isoformes de diverses protéines capillaires. Une faible couverture de séquence a empêché la distinction entre les protéines homologues de poids moléculaire similaire.

Identification of dysregulation of atrial proteins in rats with chronic obstructive apnea using two-dimensional polyacrylamide gel electrophoresis and mass spectrometry.

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.

An improved protein extraction method applied to cotton leaves is compatible with 2-DE and LC-MS.

BACKGROUND: Two-dimensional electrophoresis (2-DE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) are widely used in plant proteomics research. However, these two techniques cannot be simultaneously satisfied by traditional protein extraction methods when investigate cotton leaf proteome. RESULTS: Here, we evaluated the efficiency of three different protein extraction methods for 2-DE and LC-MS/MS analyses of total proteins obtained from cotton leaves. The protein yield of the borax/PVPP/phenol (BPP) method (0.14%) was significantly lower than the yields of the trichloroacetic acid/acetone (TCA) precipitation method (1.42%) and optimized TCA combined with BPP (TCA-B) method (0.47%). The BPP method was failed to get a clear 2-DE electrophoretogram. Fifty pairs of protein spots were randomly selected from the 2-DE gels of TCA- and TCA-B-extracted proteins for identification by MALDI TOF/TOF, and the results of 42 pairs were consistent. High-throughput proteomic analysis showed that 6339, 9282 and 9697 unique proteins were identified from the total cotton leaf proteins extracted by the TCA, BPP and TCA-B methods, respectively. Gene Ontology (GO) analysis revealed that the proteins specifically identified by TCA method were primarily distributed in the plasma membrane, while BPP and TCA-B methods specific proteins distributed in the cytosol, indicating the sub-cellular preference of different protein extraction methods. Further, ATP-dependent zinc metalloprotease FTSH 8 could be observed in the 2-DE gels of TCA and TCA-B methods, and could only be detected in the LC-MS/MS results of the BPP and TCA-B methods, showing that TCA-B method might be the optimized choice for both 2-DE and LC-MS/MS. CONCLUSION: Our data provided an improved TCA-B method for protein extraction that is compatible with 2-DE and LC-MS/MS for cotton leaves and similar plant tissues which is rich in polysaccharides and polyphenols.

Increasing the amount of phosphoric acid enhances the suitability of Bradford assay for proteomic research.

The Bradford assay is one of the most commonly used methods for protein quantification. However, in proteomic research, the lysis buffer generally used for dissolving proteins can cause some interference to the assay. The dye reagent of classical Bradford assay contains 8.50% (w/v) phosphoric acid, which is an important factor relating to the color yield of the assay. In this study, the phosphoric acid content in dye reagent was increased to 9.35% (w/v), 10.20% (w/v), and 11.05% (w/v) to evaluate the changes of interference and the effects of lysis buffer on the interaction between proteins and dye reagent. Results show that lysis buffer not only causes background interference but also affects the protein-dye chromogenic process. Analysis of different components in the lysis buffer showed that carrier ampholyte is the main factor that introduces interference to the Bradford assay. Detergents are well-known interfering compounds in the Bradford assay, but CHAPS and octyl b-D-glucopyranoside only cause slight interference. When the amount of phosphoric acid was increased from 8.50%(w/v) to 10.20% (w/v), the sensitivity of the Bradford assay to proteins in lysis buffer was increased, and the interference delivered by lysis buffer was considerably reduced.

Development of a Novel Platform of Proteome Profiling Based on an Easy-to-Handle and Informative 2D-DIGE System.

Proteome profiling based on two-dimensional (2D)-DIGE might be a useful tool for investigating drug-like compounds and the mode of action of drugs. However, obtaining data for profiling requires high labor costs, and it is difficult to control the reproducibility of spot positions because 2D-DIGE usually requires large-size glass plates and spot alignments are greatly affected by the quality of DryStrips and polyacrylamide gels (PAGs). Therefore, we have developed a novel platform by employing small size DryStrips and PAGs, and an image analysis strategy based on dual correction of spot alignment and volume. Our system can automatically detect a large number of consistent spots through all images. Cytosol fractions of HeLa cells treated with dimethyl sulfoxide (DMSO) or bortezomib were analyzed, 1697 consistent spots were detected, and 775 of them were significantly changed with the treatment. Deviations between different days and lot sets of DryStrips and PAGs were investigated by calculating the correlation coefficients. The mean values of the correlation between days and lot sets were 0.96 and 0.94, respectively. Clustering analysis of all the treatment data clearly separated the DMSO or bortezomib treated groups beyond day deviations. Thus, we have succeeded in developing an easy-to-handle 2D-DIGE system that can be a novel proteome profiling platform.