Two-dimensional Difference Gel Electrophoresis Research Articles

Comparative differential proteomic analysis of minimal change disease and focal segmental glomerulosclerosis

BackgroundMinimal change disease (MCD) and primary focal segmental glomerulosclerosis (FSGS) are glomerular diseases characterized by nephrotic syndrome. Their diagnosis requires a renal biopsy, but it is an invasive procedure with potential complications. In a small biopsy sample, where only normal glomeruli are observed, FSGS cannot be differentiated from MCD. The correct diagnosis is crucial to an effective treatment, as MCD is normally responsive to steroid therapy, whereas FSGS is usually resistant.The purpose of our study was to discover and validate novel early urinary biomarkers capable to differentiate between MCD and FSGS.MethodsForty-nine patients biopsy-diagnosed of MCD and primary FSGS were randomly subdivided into a training set (10 MCD, 11 FSGS) and a validation set (14 MCD, 14 FSGS). The urinary proteome of the training set was analyzed by two-dimensional differential gel electrophoresis coupled with mass spectrometry. The proteins identified were quantified by enzyme-linked immunosorbent assay in urine samples from the validation set.ResultsUrinary concentration of alpha-1 antitrypsin, transferrin, histatin-3 and 39S ribosomal protein L17 was decreased and calretinin was increased in FSGS compared to MCD. These proteins were used to build a decision tree capable to predict patient’s pathology.ConclusionsThis preliminary study suggests a group of urinary proteins as possible non-invasive biomarkers with potential value in the differential diagnosis of MCD and FSGS. These biomarkers would reduce the number of misdiagnoses, avoiding unnecessary or inadequate treatments.

Differential Proteome Between Patient-Related and Non-related Environmental Isolates of Legionella pneumophila.

Molecular epidemiologic studies of Legionella have shown different molecular types coexisting in the same environment, with only one having the ability to trigger an outbreak. We therefore studied the proteome of isolates of these different molecular types in search of the proteins responsible for infection. In this study, we performed a differential proteomic analysis between patient-related and non-patient-related environmental isolates using two-dimensional difference gel electrophoresis (2D-DIGE) combined with mass spectrometry. Sixty-three spots were observed as being different between the two groups; 31 spots were identified corresponding to 23 different proteins. Patient-related isolates overexpressed proteins associated with metabolism, with enzymes of the tricarboxylic acid cycle and the degradation pathways being the most abundant proteins identified. However, the largest group of non-patient-related proteins was associated with stress response. Furthermore, the MOMP protein was located in different spots depending on their patient-related or non-patient-related origin, suggesting different post-translational modifications. According to these results, different bacterial adaptation pathways are activated in stress conditions which influence their ability to produce infection.

Plasma protein profiling for potential biomarkers in the early diagnosis of Alzheimer’s disease

Objectives: Alzheimer’s disease (AD) is the most common cause of dementia in elderly persons. Since the pathology of AD develops slowly from a preclinical or early phase into a fully expressed clinical syndrome, at the time of diagnosis the disease has been progressing for many years. To facilitate the early diagnosis of AD, we performed protein profiling of blood in patients with mild AD as defined by the Functional Assessment Staging (FAST) scale.Methods: Plasma samples from mild AD patients and healthy controls were analyzed using two-dimensional differential gel electrophoresis (2D-DIGE) combined with matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry (MALDI-TOF/TOF/MS) followed by peptide mass fingerprinting.Results: Three downregulated proteins were identified: apolipoprotein A-1, alpha-2-HS-glycoprotein, and afamin. Two proteins, including apolipoprotein A-4 and fibrinogen gamma chain, were upregulated in mild AD patients.Discussion: Our results suggest that altered expression levels of these proteins in plasma may yield candidate biomarkers for the early diagnosis of AD.Abbreviations: AD, Alzheimer’s disease; FAST, Functional Assessment Staging; 2D-DIGE, two-dimensional differential gel electrophoresis; MALDI-TOF/TOF/MS, matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry; CSF, cerebrospinal fluid; Aβ, amyloid beta; MMSE, Mini Mental State Examination; MRI, magnetic resonance imaging; NINCDS-ADRDA, National Institute for Neurological Diseases and Stroke/Alzheimer’s Disease and Related Disorders Association; CHAPS, 3-((3-cholamidopropyl) dimethylammonio)-1-propanesulfonate; DTT, dithiothreitol; SDS-PAGE, SDS-polyacrylamide gel electrophoresis; DIA, differential in-gel analysis; BVA, biological variation analysis; CBB, Coomassie brilliant blue; 2DE, two-dimensional gel electrophoresis; TFA, trifluoroacetic acid; ACTH, adrenocorticotropic hormone; Apo A-1, apolipoprotein A-1; AHSG, alpha-2-HS-glycoprotein; Apo A-4, apolipoprotein A-4; MCI, mild cognitive impairment.

The short- and long-term proteomic effects of sleep deprivation on the cortical and thalamic synapses.

Acute total sleep deprivation (SD) impairs memory consolidation, attention, working memory and perception. Structural, electrophysiological and molecular experimental approaches provided evidences for the involvement of sleep in synaptic functions. Despite the wide scientific interest on the effects of sleep on the synapse, there is a lack of systematic investigation of sleep-related changes in the synaptic proteome. We isolated parietal cortical and thalamic synaptosomes of rats after 8h of total SD by gentle handling and 16h after the end of deprivation to investigate the short- and longer-term effects of SD on the synaptic proteome, respectively. The SD efficiency was verified by electrophysiology. Protein abundance alterations of the synaptosomes were analyzed by fluorescent two-dimensional differential gel electrophoresis and by tandem mass spectrometry. As several altered proteins were found to be involved in synaptic strength regulation, our data can support the synaptic homeostasis hypothesis function of sleep and highlight the long-term influence of SD after the recovery sleep period, mostly on cortical synapses. Furthermore, the large-scale and brain area-specific protein network change in the synapses may support both ideas of sleep-related synaptogenesis and molecular maintenance and reorganization in normal rat brain.

Determination of Protein Markers for Beef Tenderness in U.S. Select Beef

ObjectivesBeef tenderness is a primary quality feature that defines the consumer’s perception of the product quality. Therefore definition of chemical characteristics that contribute to variation in tenderness within a specific USDA grade can help refine our understanding of meat tenderness. The objective of this experiment was to identify novel protein markers for tenderness in Longissimus dorsi sarcoplasmic fractions utilizing proteomics and a valuable library of beef samples. Materials and MethodsSteaks (USDA Select grade) used in this trial were classified as low shear force (LSF, n = 12; 11.3 ± 1.6 kg) and high shear force (HSF, n = 12; 34.3 ± 4.7 kg), according to their slice shear force (SSF, kg) measurements at 14 d postmortem. Western blotting of whole-muscle samples was used to determine troponin-T degradation (indicator of overall proteolysis). Sarcoplasmic protein samples from d 2 postmortem were prepared for two-dimensional difference in gel electrophoresis (2D-DIGE). Samples from LSF and HSF were sorted according to SSF values and were matched to a pooled internal reference (representative of all samples). Protein samples were loaded (75 µg) on 24 cm strips, pH 6 to 9, and focused in immobilized pH gradient in the first dimension. For the second dimension the strips were run on 12.5% acrylamide gels (n = 12). After 2D-DIGE, protein spots found to be different in relative abundance between HSF and LSF samples, and with other proteins of interest, were identified using Q Exactive Hybrid Quadrupole-Orbitrap Mass Spectrometer (LC-MS/MS). Immunoblots were performed to confirm protein identity. ResultsThe LSF samples exhibited greater troponin-T degradation (as evidenced by abundance of a 30 kDa fragment) than the HSF samples (LSF ratio to reference was 1.8, HSF ratio to reference was < 0.01; P < 0.01). A total of 31 spots showed a significant difference (P < 0.1) in relative abundance across groups (HSF vs. LSF). Among these, 18 spots were identified as 8 different proteins (Table 1). Proteins that were more abundant in the LSF samples are metabolic enzymes involved in the glycolytic pathway (glyceraldehyde-3-phosphate dehydrogenase, fructose bisphosphate aldolase A). Among the six proteins that showed greater abundance in the HSF samples, four belong to the oxidative energy metabolism pathways (malate dehydrogenase, creatine kinase M-type, myoglobin, carbonic anhydrase 3), one is involved in redox pathways (peroxiredoxin-1) and another one in protein turnover (polyubiquitin). The greater abundance of spots corresponding to oxidative metabolism enzymes suggests that aerobic metabolism may decrease potential for postmortem proteolysis. Table 1Differentially abundant proteins in high and low slice shear force groups Protein% CoverageRatio (LSF/HSF)P-valueGlyceraldehyde-3-phosphate dehydrogenase (GAPDH)26.731.980.060Fructose-bisphosphate aldolase A62.361.79< 0.050Polyubiquitin (Fragment)21.22–1.180.100Malate dehydrogenase, mitochondrial63.31–1.18< 0.050Creatine kinase M-type70.87–1.190.080Myoglobin99.35–1.290.095Carbonic anhydrase 379.12–1.56< 0.010Perociredoxin-170.85–1.240.055ConclusionThe results demonstrate that variation in tenderness within U.S. Select Top loin steaks is due in large part to variation in proteolysis. The variation in tenderness and proteolysis in these defined groups may be partially explained by a greater abundance of metabolic proteins involved in oxidative metabolism. This project was funded in part by grants from the Beef Checkoff (Denver, CO) and the Iowa Beef Industry Council (Ames, IA). The scholarship for the first author was granted by CNPq-Brazil.

Determination of Protein Markers for Beef Tenderness in U.S. Select Beef

ObjectivesBeef tenderness is a primary quality feature that defines the consumer’s perception of the product quality. Therefore definition of chemical characteristics that contribute to variation in tenderness within a specific USDA grade can help refine our understanding of meat tenderness. The objective of this experiment was to identify novel protein markers for tenderness in Longissimus dorsi sarcoplasmic fractions utilizing proteomics and a valuable library of beef samples. Materials and MethodsSteaks (USDA Select grade) used in this trial were classified as low shear force (LSF, n = 12; 11.3 ± 1.6 kg) and high shear force (HSF, n = 12; 34.3 ± 4.7 kg), according to their slice shear force (SSF, kg) measurements at 14 d postmortem. Western blotting of whole-muscle samples was used to determine troponin-T degradation (indicator of overall proteolysis). Sarcoplasmic protein samples from d 2 postmortem were prepared for two-dimensional difference in gel electrophoresis (2D-DIGE). Samples from LSF and HSF were sorted according to SSF values and were matched to a pooled internal reference (representative of all samples). Protein samples were loaded (75 µg) on 24 cm strips, pH 6 to 9, and focused in immobilized pH gradient in the first dimension. For the second dimension the strips were run on 12.5% acrylamide gels (n = 12). After 2D-DIGE, protein spots found to be different in relative abundance between HSF and LSF samples, and with other proteins of interest, were identified using Q Exactive Hybrid Quadrupole-Orbitrap Mass Spectrometer (LC-MS/MS). Immunoblots were performed to confirm protein identity. ResultsThe LSF samples exhibited greater troponin-T degradation (as evidenced by abundance of a 30 kDa fragment) than the HSF samples (LSF ratio to reference was 1.8, HSF ratio to reference was < 0.01; P < 0.01). A total of 31 spots showed a significant difference (P < 0.1) in relative abundance across groups (HSF vs. LSF). Among these, 18 spots were identified as 8 different proteins (Table 1). Proteins that were more abundant in the LSF samples are metabolic enzymes involved in the glycolytic pathway (glyceraldehyde-3-phosphate dehydrogenase, fructose bisphosphate aldolase A). Among the six proteins that showed greater abundance in the HSF samples, four belong to the oxidative energy metabolism pathways (malate dehydrogenase, creatine kinase M-type, myoglobin, carbonic anhydrase 3), one is involved in redox pathways (peroxiredoxin-1) and another one in protein turnover (polyubiquitin). The greater abundance of spots corresponding to oxidative metabolism enzymes suggests that aerobic metabolism may decrease potential for postmortem proteolysis. Table 1Differentially abundant proteins in high and low slice shear force groups Protein% CoverageRatio (LSF/HSF)P-valueGlyceraldehyde-3-phosphate dehydrogenase (GAPDH)26.731.980.060Fructose-bisphosphate aldolase A62.361.79< 0.050Polyubiquitin (Fragment)21.22–1.180.100Malate dehydrogenase, mitochondrial63.31–1.18< 0.050Creatine kinase M-type70.87–1.190.080Myoglobin99.35–1.290.095Carbonic anhydrase 379.12–1.56< 0.010Perociredoxin-170.85–1.240.055ConclusionThe results demonstrate that variation in tenderness within U.S. Select Top loin steaks is due in large part to variation in proteolysis. The variation in tenderness and proteolysis in these defined groups may be partially explained by a greater abundance of metabolic proteins involved in oxidative metabolism. This project was funded in part by grants from the Beef Checkoff (Denver, CO) and the Iowa Beef Industry Council (Ames, IA). The scholarship for the first author was granted by CNPq-Brazil.

A Two-Dimensional Difference Gel Electrophoresis (2D-DIGE) Protocol for Studies of Neural Precursor Cells.

This chapter describes the basics of two-dimensional difference gel electrophoresis (2D-DIGE) for multiplex analysis of up to distinct proteomes. The example given describes the analysis of undifferentiated and differentiated neural precursor cells labelled with fluorescent Cy3 and Cy5 dyes in comparison to a pooled standard labelled with Cy2. After labelling, the proteomes are mixed together and electrophoresed on the same 2D gels. Scanning the gels at wavelengths specific for each dye allows direct overlay of the two different proteomes and the differences in abundance of specific protein spots can be determined through comparison to the pooled standard.

2D-DIGE in Proteomics.

The two-dimensional difference gel electrophoresis method is a valuable approach for proteomics. The method, using cyanine fluorescent dyes, allows the co-migration of multiple protein samples in the same gel and their simultaneous detection, thus reducing experimental and analytical time. 2D-DIGE, compared to traditional post-staining 2D-PAGE protocols (e.g., colloidal Coomassie or silver nitrate), provides faster and more reliable gel matching, limiting the impact of gel to gel variation, and allows also a good dynamic range for quantitative comparisons. By the use of internal standards, it is possible to normalize for experimental variations in spot intensities and gel patterns. Here we describe the experimental steps we follow in our routine 2D-DIGE procedure that we then apply to multiple biological questions.

Growth of Murine Splenic Tissue Is Suppressed by Lymphotoxin β-Receptor Signaling (LTβR) Originating from Splenic and Non-Splenic Tissues.

Development and maintenance of secondary lymphoid organs such as lymph nodes and spleen essentially depend on lymphotoxin β-receptor (LTβR) signaling. It is unclear, however, by which molecular mechanism their size is limited. Here, we investigate whether the LTβR pathway is also growth suppressing. By using splenic tissue transplantation it is possible to analyze a potential contribution of LTβR signaling inside and outside of the implanted tissue. We show that LTβR signaling within the endogenous spleen and within non-splenic tissues both significantly suppressed the regeneration of implanted splenic tissue. The suppressive activity positively correlated with the total number of LTβR expressing cells in the animal (regenerate weights of 115 ± 8 mg in LTβR deficient recipients and of 12 ± 9 mg in wild-type recipients), affected also developed splenic tissue, and was induced but not executed via LTβR signaling. Two-dimensional differential gel electrophoresis and subsequent mass spectrometry of stromal splenic tissue was applied to screen for potential factors mediating the LTβR dependent suppressive activity. Thus, LTβR dependent growth suppression is involved in regulating the size of secondary lymphoid organs, and might be therapeutically used to eradicate tertiary lymphoid tissues during autoimmune diseases.

Global Identification of ERK Substrates by Phosphoproteomics Based on IMAC and 2D-DIGE.

Extracellular signal-regulated kinase (ERK) regulates various cellular functions through phosphorylation of numerous downstream substrates, which have not yet been fully characterized. To date, several phosphoproteomic approaches have been employed to identify novel substrates for ERK. In this chapter, we describe a method to globally identify ERK substrates by combining immobilized metal affinity chromatography (IMAC) and two-dimensional difference gel electrophoresis (2D-DIGE) followed by mass spectrometry. Phosphoprotein enrichment by IMAC enables the subsequent detection of many protein spots with different fluorescence intensities between ERK-inhibited and -activated cells in 2D-DIGE analysis. Furthermore, the advanced sensitivity and resolution of liquid chromatography coupled with tandem mass spectrometry allow for a direct identification of proteins obtained from silver-stained 2D-DIGE gels. Validation experiments such as Phos-tag Western blotting are important steps to further elucidate the functional roles of ERK-mediated phosphorylation of these newly identified substrates.

Label Free Quantitative Mass Spectrometry Identifies Processes Linked to Platelet Degranulation As Early Events during Platelet Storage

BackgroundPlatelet concentrates (PCs) are stored at room temperature to preserve their biological activity. To minimize the risk of bacterial outgrowth, storage time is limited to 7 days. It is well-established that prolonged storage of PCs results in modifications that result in a decreased hemostatic efficacy. This loss of platelet functionality during storage is commonly referred to as the platelet storage lesion (PSL). Typical events linked to development of the PSL are shape changes, platelet activation and loss of receptors crucial for platelet functionality. Two-dimensional (2D) differential gel electrophoresis (DIGE), isotope tagging and isotope-coded affinity tagging (ICAT) have been previously used to monitor changes in protein composition during storage. These studies have provided valuable insights into the changes associated with the PSL, however, these studies generally focused on a limited set of proteins.AimWe aimed to generate an overview of changes in the platelet proteome during storage using label free quantitative mass spectrometry. Furthermore, we employed Gene ontology (GO) enrichment analysis to identify pathways and biological processes that were linked to development of the PSL.MethodsThree independently pooled PCs were stored in plasma under standard blood bank conditions for 16 days. Tryptic peptides were separated by nanoscale C18 reverse phase chromatography coupled on line to an Orbitrap Fusion Tribrid mass spectrometer. The RAW mass spectrometry files were processed with the MaxQuant computational platform. The global changes in protein level during platelet storage were assessed employing the analysis-of-variance functions of PERSEUS. Gene ontology enrichment analysis of biological processes, molecular functions and cellular compartments of the significantly different proteins was performed using the Cytoscape plug-in BiNGO.ResultsA total number of 2501 proteins was detected in all 3 biological replicates in at least one of the time points analyzed. The analysis showed that 18 proteins were down-regulated over time, whereas the level of 3 proteins was found to increase. CytoScape BinGo analysis of these significantly downregulated proteins revealed that the majority of this set was linked to GO-terms platelet degranulation, secretion and regulated exocytosis. This set of proteins included von Willebrand factor (VWF), serglycin (SRGN), SPARC, amyloid beta A4 protein (APP), multimerin-1 (MMRN1) and platelet factor 4 (PF4). A significant decline in these protein levels was observed at day 5 of storage, suggesting that release of α-granules is a relatively early event during platelet storage. At day 5 also a marked decline in S100A9 was observed. S100A9 has been implicated in degranulation in neutrophils, and may therefore also be linked to platelet granule release. Levels of membrane surface platelet glycoproteins such as glycoprotein Ibα did not significantly change at day 5. Only one single protein, histone H2A, was found to be consistently decreased already at two days of storage, but the significance of this finding is not clear. Upon prolonged storage (13 and 16 days) an increase in the level of α-2-macroglobulin (A2M), immunoglobulin M (IGM) and glycogenin-1 (GYG1) was observed suggesting that platelets acquire an (increased) potential to bind and/or internalize proteins from their environment. Consistent with this notion we also detected significant levels of several serine protease inhibitors, although levels of these proteins did not change upon storage.ConclusionsOverall, our findings highlight dynamic changes in protein composition of platelets during storage. Our data provide evidence for sustained release of α-granules over time which becomes significant at day 5. Our data also suggest that during storage, platelets can bind or ingest proteins from their environment which may have impact on the hemostatic properties of stored platelets. DisclosuresLeebeek:CSL Behring: Membership on an entity's Board of Directors or advisory committees, Research Funding; Baxalta: Consultancy, Membership on an entity's Board of Directors or advisory committees; Dutch Hemphilia Foundation: Research Funding.

Simultaneous Two-Dimensional Difference Gel Electrophoresis (2D-DIGE) Analysis of Two Distinct Proteomes.

This chapter describes the basics, applications, and limitations of two-dimensional gel electrophoresis (2DE) and two-dimensional difference gel electrophoresis (2D-DIGE) for multiplex analysis of distinct proteomes. We also propose a basic protocol for 2D-DIGE, technique that allows the analysis of paired protein extracts, which are labeled with fluorescent Cy3 and Cy5 dyes and electrophoresed with a Cy2-labeled standard extract on the same 2DE gels. Scanning the gels at wavelengths specific for each dye allows direct overlay the two different proteomes and the differences in abundance of specific protein spots can be determined.

Proteomics Identification of Potential Candidates Involved in Cell Proliferation for Early Stage of Brain Regeneration in the Adult Zebrafish.

The central nervous system (CNS) of the non-mammalian vertebrates has better neuroregenerative capability as compared with the mammalian CNS. Regeneration of habenula was observed 40 days after damage in zebrafish. During the early stage of regeneration, we found a significant increase of apoptotic cells on day-1 post-damage and of proliferative cells on day-3 post-damage. To identify the molecular factor(s) involved in the early stages of neuroregeneration, differentially expressed proteins during sham, 20- and 40-h post-habenula damage were investigated by proteomic approach by using two-dimensional differential gel electrophoresis (2D-DIGE) coupled with Matrix-Assisted Laser Desorption/Ionization-Time-of-Flight (MALDI-ToF) and tandem mass spectrometry. Protein profiles revealed 17 differentially (>1.5-fold) expressed proteins: 10 upregulated, 4 downregulated, 2 proteins were found to be downregulated at the early stage but upregulated at a later stage, and 1 protein was found to be upregulated at 2 different time points. All proteins identified can be summarized under few molecular processes involved in the early stages of neuroregeneration in zebrafish CNS: apoptosis regulation (Wnt inhibitory factor 1 [WIF1]), neuroprotection (metallothionein), cell proliferation (Spred2, ependymin, Lhx1, and Wnts), differentiation (Spred2, Lhx9, and Wnts), and morphogenesis (cytoplasmic actins and draculin). These protein profiling results suggest that drastic molecular changes occur in the neuroregenerative process during this period, which includes cell proliferation, differentiation, and protection.

Cytoskeletal abnormalities and neutrophil dysfunction in WDR1 deficiency

AbstractCell motility, division, and structural integrity depend on dynamic remodeling of the cellular cytoskeleton, which is regulated in part by actin polymerization and depolymerization. In 3 families, we identified 4 children with recurrent infections and varying clinical manifestations including mild neutropenia, impaired wound healing, severe stomatitis with oral stenosis, and death. All patients studied had similar distinctive neutrophil herniation of the nuclear lobes and agranular regions within the cytosol. Chemotaxis and chemokinesis were markedly impaired, but staphylococcal killing was normal, and neutrophil oxidative burst was increased both basally and on stimulation. Neutrophil spreading on glass and cell polarization were also impaired. Neutrophil F-actin was elevated fourfold, suggesting an abnormality in F-actin regulation. Two-dimensional differential in-gel electrophoresis identified abnormal actin-interacting protein 1 (Aip1), encoded by WDR1, in patient samples. Biallelic mutations in WDR1 affecting distinct antiparallel β-strands of Aip1 were identified in all patients. It has been previously reported that Aip1 regulates cofilin-mediated actin depolymerization, which is required for normal neutrophil function. Heterozygous mutations in clinically normal relatives confirmed that WDR1 deficiency is autosomal recessive. Allogeneic stem cell transplantation corrected the immunologic defect in 1 patient. Mutations in WDR1 affect neutrophil morphology, motility, and function, causing a novel primary immunodeficiency.

Up-Regulation of Antioxidant Proteins in the Plasma Proteome during Saturation Diving: Unique Coincidence under Hypobaric Hypoxia

Saturation diving (SD) is one of the safest techniques for tolerating hyperbaric conditions for long durations. However, the changes in the human plasma protein profile that occur during SD are unknown. To identify differential protein expression during or after SD, 65 blood samples from 15 healthy Japanese men trained in SD were analyzed by two-dimensional fluorescence difference gel electrophoresis. The expression of two proteins, one 32.4 kDa with an isoelectric point (pI) of 5.8 and the other 44.8 kDa with pI 4.0, were elevated during SD to 60, 100, and 200 meters sea water (msw). The expression of these proteins returned to pre-diving level when the SD training was completed. The two proteins were identified using in-gel digestion and mass spectrometric analysis; the 32.4 kDa protein was transthyretin and the 44.8 kDa protein was alpha-1-acid glycoprotein 1. Oxidation was detected at methionine 13 of transthyretin and at methionine 129 of alpha-1-acid glycoprotein 1 by tandem mass spectrometry. Moreover, haptoglobin was up-regulated during the decompression phase of 200 msw. These plasma proteins up-regulated during SD have a common function as anti-oxidants. This suggests that by coordinating their biological effects, these proteins activate a defense mechanism to counteract the effects of hyperbaric-hyperoxic conditions during SD.

Modulation of plant growth in vivo and identification of kinase substrates using an analog-sensitive variant of CYCLIN-DEPENDENT KINASE A;1.

BackgroundModulation of protein activity by phosphorylation through kinases and subsequent de-phosphorylation by phosphatases is one of the most prominent cellular control mechanisms. Thus, identification of kinase substrates is pivotal for the understanding of many – if not all – molecular biological processes. Equally, the possibility to deliberately tune kinase activity is of great value to analyze the biological process controlled by a particular kinase.ResultsHere we have applied a chemical genetic approach and generated an analog-sensitive version of CDKA;1, the central cell-cycle regulator in Arabidopsis and homolog of the yeast Cdc2/CDC28 kinases. This variant could largely rescue a cdka;1 mutant and is biochemically active, albeit less than the wild type. Applying bulky kinase inhibitors allowed the reduction of kinase activity in an organismic context in vivo and the modulation of plant growth. To isolate CDK substrates, we have adopted a two-dimensional differential gel electrophoresis strategy, and searched for proteins that showed mobility changes in fluorescently labeled extracts from plants expressing the analog-sensitive version of CDKA;1 with and without adding a bulky ATP variant. A pilot set of five proteins involved in a range of different processes could be confirmed in independent kinase assays to be phosphorylated by CDKA;1 approving the applicability of the here-developed method to identify substrates.ConclusionThe here presented generation of an analog-sensitive CDKA;1 version is functional and represent a novel tool to modulate kinase activity in vivo and identify kinase substrates. Our here performed pilot screen led to the identification of CDK targets that link cell proliferation control to sugar metabolism, proline proteolysis, and glucosinolate production providing a hint how cell proliferation and growth are integrated with plant development and physiology.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-016-0900-7) contains supplementary material, which is available to authorized users.

Patients with calcific aortic stenosis exhibit systemic molecular evidence of ischemia, enhanced coagulation, oxidative stress and impaired cholesterol transport

BackgroundThe most common valve diseases are calcific aortic stenosis (AS) and aortic regurgitation (AR). The former is characterized by thickening of valve leaflets followed by progressive calcification, which produces progressive aortic valve (AV) narrowing, increased pressure afterload on the left ventricle (LV) and subsequent LV hypertrophy. On the other hand, AR is due to malcoaptation of the valve leaflets with resultant diastolic reflux of blood from aorta back to the LV producing volume and pressure overload and progressive LV dilatation. In order to isolate the molecular mechanisms taking place during AS, we have used an integrated “–omic” approach to compare plasma samples from AS and from AR patients used as controls. The final purpose of this work is to find molecular changes in response to the calcification of the AV, diminishing the effects of the AV dysfunction. Methods and resultsUsing two-dimensional difference gel electrophoresis (2D-DIGE) and gas chromatography coupled to mass spectrometry (GC-MS) in a cohort of 6 subjects, we have found differences in 24 protein spots and 19 metabolites, respectively. Among them, 7 proteins and 3 metabolites have been verificated by orthogonal techniques (SRM or turbidimetry): fibrinogen beta and gamma chain, vitronectin, apolipoprotein C-II, antithrombin III, haptoglobin, succinic acid, pyroglutamic acid and alanine. Classification according to their main function showed alterations related to coagulation, inflammation, oxidative stress, response to ischemia and lipid metabolism, defining 4 different molecular panels that characterize AS with high specificity and sensitivity. ConclusionThese results may facilitate management of these patients by making faster diagnostics of the disease and better understand these pathways for regulating its progression.

Seedling development in maize cv. B73 and blue light-mediated proteomic changes in the tip vs. stem of the coleoptile.

In 2009, the draft genome of the reference inbred line of maize (Zea mays L. spp. mays cv. B73) was published so that, using this specific corn variety, molecular analyses of physiological processes became possible. However, the morphology and developmental patterns of B73 maize, compared with that of the more frequently used hybrid varieties, have not yet been analyzed. Here, we describe organ development in seedlings of B73 maize and in those of six other hybrid cultivars, and document significant morphological as well as quantitative differences between these varieties of Z. mays. In a second set of experiments, we used etiolated seedlings of B73 maize to analyze the effect of blue light (BL) on the patterns of proteins in the tip vs. growing region of this sheath-like organ. By using two-dimensional difference gel electrophoresis (2D DIGE), coupled with tandem mass spectrometry, we detected, in the microsomal fraction of maize coleoptile tips, rapid changes in the abundance of protein spots of maize phototropin 1 and several metabolic enzymes. In the sub-apical (growing) region of the coleoptile, proteomic changes were less pronounced. These results suggest that the tip of the coleoptile of B73 maize may serve as a unique model system for dissecting BL responses in a light-sensitive plant organ of known function.

Biosynthesis and Regulation of Wheat Amylose and Amylopectin from Proteomic and Phosphoproteomic Characterization of Granule-binding Proteins.

Waxy starch has an important influence on the qualities of breads. Generally, grain weight and yield in waxy wheat (Triticum aestivum L.) are significantly lower than in bread wheat. In this study, we performed the first proteomic and phosphoproteomic analyses of starch granule-binding proteins by comparing the waxy wheat cultivar Shannong 119 and the bread wheat cultivar Nongda 5181. These results indicate that reduced amylose content does not affect amylopectin synthesis, but it causes significant reduction of total starch biosynthesis, grain size, weight and grain yield. Two-dimensional differential in-gel electrophoresis identified 40 differentially expressed protein (DEP) spots in waxy and non-waxy wheats, which belonged mainly to starch synthase (SS) I, SS IIa and granule-bound SS I. Most DEPs involved in amylopectin synthesis showed a similar expression pattern during grain development, suggesting relatively independent amylose and amylopectin synthesis pathways. Phosphoproteome analysis of starch granule-binding proteins, using TiO2 microcolumns and LC-MS/MS, showed that the total number of phosphoproteins and their phosphorylation levels in ND5181 were significantly higher than in SN119, but proteins controlling amylopectin synthesis had similar phosphorylation levels. Our results revealed the lack of amylose did not affect the expression and phosphorylation of the starch granule-binding proteins involved in amylopectin biosynthesis.

Proteomic and network analysis of pregnancy-induced changes in the porcine endometrium on Day 12 of gestation.

Conceptus attachment is a time-sensitive process that requires a synchronized uterine environment created by molecular changes in the endometrium in response to ovarian hormones and conceptus signals. Porcine conceptuses undergo rapid elongation and differentiation, and secrete estrogens that serve as maternal-recognition-of-pregnancy signals during the peri-implantation period (Days 11-12). Pregnancy-induced proteomic changes in the porcine endometrium were measured during this period using two-dimensional differential gel electrophoresis of endometrial protein lysates from Day-12 pregnant versus non-pregnant animals (n = 4 each). Forty-four differentially abundant proteins in the pregnant endometrium were identified by mass spectrometry. The pregnant endometrium was associated with a unique protein profile, revealed by principal component analysis. A pregnancy-dependent increase in the abundance of serpins, cofilin, annexin A2, aldose reductase, cyclophilin, protein disulphide isomerase A3, and peroxiredoxin 1 was observed. Western blotting for some of the selected proteins confirmed their enrichment during pregnancy. Ingenuity pathway analysis identified several functions specifically over-represented among the differentially abundant proteins in the pregnant endometrium, including calcium signaling, angiogenesis, leukocyte migration, and cell movement. Interleukin-1 beta and beta-estradiol were identified as upstream regulators of several high-abundance proteins from pregnancy. Therefore, signals from porcine conceptuses, such as estrogens, interleukin 1B, and epidermal growth factor, either alone or in coordination with other factors, prepare the uterus for implantation. Mol. Reprod. Dev. 83: 827-841, 2016 © 2016 Wiley Periodicals, Inc.