Simultaneous immunoassay of phosphorylated proteins based on apoferritin templated metallic phosphates as voltammetrically distinguishable signal reporters

2016 ◽  
Vol 80 ◽  
pp. 201-207 ◽  
Author(s):  
Xiaoxiao Ge ◽  
Aidong Zhang ◽  
Yuehe Lin ◽  
Dan Du
Keyword(s):  
Phosphorylated Proteins ◽  
Simultaneous Immunoassay

Co-Precipitation of Phosphorylated Proteins Using Trivalent Cerium-, Holmium-, and Thulium Cations

2014 ◽  
Vol 10 (3) ◽  
pp. 175-184 ◽  
Author(s):  
Matthias Rainer ◽  
Yuksel Guzel ◽  
Christoph Messner ◽  
Gunther Karl Bonn
Keyword(s):  
Phosphorylated Proteins ◽  
Trivalent Cerium ◽  
Co Precipitation

scholarly journals Transcriptome-(phospho)proteome characterization of brain of a germline model of cytoplasmic-predominant Pten expression with autism-like phenotypes

2021 ◽  
Vol 6 (1) ◽  
Author(s):  
Stetson Thacker ◽  
Charis Eng
Keyword(s):  
Autism Spectrum ◽  
Pten Expression ◽  
Differentially Expressed ◽  
Risk Genes ◽  
Animal Modeling ◽  
Network Analyses ◽  
Phosphorylated Proteins ◽  
Mass Spectrometry Technology ◽  
Neurological Processes

AbstractPTEN has a strong Mendelian association with autism spectrum disorder (ASD), representing a special case in autism’s complex genetic architecture. Animal modeling for constitutional Pten mutation creates an opportunity to study how disruption of Pten affects neurobiology and glean potential insight into ASD pathogenesis. Subsequently, we comprehensively characterized the neural (phospho)proteome of Ptenm3m4/m3m4 mice, which exhibits cytoplasmic-predominant Pten expression, by applying mass spectrometry technology to their brains at two-weeks- (P14) and six-weeks-of-age (P40). The differentially expressed/phosphorylated proteins were subjected to gene enrichment, pathway, and network analyses to assess the affected biology. We identified numerous differentially expressed/phosphorylated proteins, finding greater dysregulation at P40 consistent with prior transcriptomic data. The affected pathways were largely related to PTEN function or neurological processes, while scant direct overlap was found across datasets. Network analysis pointed to ASD risk genes like Pten and Psd-95 as major regulatory hubs, suggesting they likely contribute to initiation or maintenance of cellular and perhaps organismal phenotypes related to ASD.

scholarly journals Weight changes in hypertensive patients with phlegm-dampness syndrome: an integrated proteomics and metabolomics approach

2021 ◽  
Vol 16 (1) ◽  
Author(s):  
Chi Zhang ◽  
Li Li ◽  
Shiping Cheng ◽  
Debajyoti Chowdhury ◽  
Yong Tan ◽  
...  
Keyword(s):  
Pathway Analysis ◽  
Complement C3 ◽  
Integrative Approach ◽  
Weight Changes ◽  
Serum Samples ◽  
Phosphorylated Proteins ◽  
Circulating Markers ◽  
One Year ◽  
Study Participants

Abstract Background Hypertension (HTN) patients who have phlegm-dampness syndrome (PDS) tend to be obese and have worse outcomes. However, the association of body weight (BW) changes and mechanisms underlying the pathophysiology of HTN-PDS are not well elucidated. This study aims to identify the longitudinal observations associated with the circulating markers discriminating BW changes of individuals with HTN-PDS. Methods An integrative approach relying on metabolomics and proteomics was applied to serum samples from HTN-PDS patients in a prospective cohort to identify the plausible mechanistic pathways underpinning HTN-PDS pathophysiology. Study participants were determined to have experienced a weight change if they showed a 5%–15% increase/reduction in BW at the end of the follow-up period. The joint pathway analysis and network analysis were performed using Ingenuity Pathway Analysis (IPA®) on the serum samples obtained from the participants over the period. Results The study involved 22 HTN-PDS patients who were overweight initially and were able to lose enough weight and 24 HTN-PDS individuals who developed overweight from normal BMI during a one-year follow-up. Our analysis suggested three types of phosphatidylcholine (PC) were altered. PC (22:2(13Z,16Z)/24:1(15Z)) and LysoPC (16:1(9Z)) were decreased in Queryweight gain samples, whereas the levels of PC (14:0/16:0) were increased in weight loss samples. The metabolomic analysis suggested 24 metabolites associated with HTN-PDS. Of them, 13 were up-regulated and 11 were down-regulated. The two-dimensional difference gel electrophoresis (2D DIGE) identified 45 phosphorylated proteins got altered in the HTN-PDS patients, wherein 23 were up-regulated and 22 were down-regulated. Integrated proteomic and metabolomics analyse acknowledged biomarkers PC, Complement C3, C4a/C4b, A2M and SERPINF1 as strong predictors for BW changes in HTN-PDS patients. Conclusion The combined serum proteomic and metabolomic profiling reveals a link between BW change and the complement system activity, altered phosphatidylcholine metabolism in HTN-PDS patients. Future studies with larger cohorts are required to strengthen and validate these findings.

scholarly journals P013 The JAK-3 and TYK-2 / STAT pathways are activated in moderate to severe ulcerative colitis

2020 ◽  
Vol 14 (Supplement_1) ◽  
pp. S136-S137
Author(s):  
M Loza ◽  
J M Brea ◽  
C Calviño-Suarez ◽  
I Baston-Rey ◽  
R Ferreiro-Iglesias ◽  
...  
Keyword(s):  
Ulcerative Colitis ◽  
Animal Models ◽  
Inflammatory Response ◽  
Western Blot ◽  
Signalling Pathways ◽  
Target Engagement ◽  
Inclusion Criteria ◽  
Phosphorylated Proteins ◽  
Single Centre Study ◽  
Stat Pathway

Abstract Background Ulcerative colitis (UC) is a chronic, progressive and disabling disease with a complex pathology of unknown aetiology influenced by genetic, environmental and microbiota factors that lead to an immunological and inflammatory response in the colon. Janus Activated Kinase (JAK) family plays a key role in modulating the adaptive and innate inflammatory response. The JAK/STAT pathway involvement in UC has been demonstrated in both animal models and human studies. Thus, overexpressed JAK-3 has been detected in the intestine of patients with UC, suggesting a key role in their pathophysiology and the inhibition of TYK-2 in animal models resulted in an improvement of the disease, which would explain its implication in the inflammatory process. We hypothesise here that there could be an activation of JAK-3 and TYK-2 signalling pathways in UC patients. Thus, we aimed to detect the activation of both signalling pathways by means of western-blot studies in UC patient samples Methods A prospective, observational single-centre study was designed. Inclusion criteria were adult patients with endoscopic active UC (more than Mayo-0) confirmed in a programmed colonoscopy. All patients signed informed consent. Samples were obtained from overstock of routine biopsies in the more severe segment affected of the large bowel. Tissues were homogenised and processed in order to obtain cell lysates by employing RIPA buffer and ultrasounds. The degree of activation of the JAK-3 and TYK-2 pathways was measured by detecting the phosphorylation of both targets as well as of STAT1, STAT3, STAT4, STAT5 and STAT6 through western blot by employing specific antibodies for total and phosphorylated proteins. Results 19 UC patients were consecutively included. Mean age was 46 years old. 53% were female, 47% were extensive colitis (E3) and 53% left-side colitis (E2). Regarding endoscopic activity, 26% had Mayo-1, 53% Mayo-2, and 21% Mayo-3. Immunoreactive bands for both phosphorylated JAK-3 and TYK-2 were detected in the biopsies from UC patients, evidencing that colonic inflammation leads to an activation of both targets. The study of STATs phosphorylation showed immunoreactive bands for phosphorylated forms of STAT1, STAT3, STAT4, STAT5 and STAT6 confirming the activation of both signalling-pathways in these patients (Figure 1). Conclusion The developed translational workflows involving basic/clinical research confirm the activation of both JAK-3 and TYK-2-dependent signalling pathways in UC patients, validating both kinases as targets for treating UC. The developed methodology allows studying the target engagement for future JAK-3/ TYK-2 inhibitors employed in clinical trials.

scholarly journals Studies on the incorporation of [32P]phosphate into pyruvate dehydrogenase in intact rat fat-cells. Effects of insulin

1980 ◽  
Vol 192 (2) ◽  
pp. 469-481 ◽  
Author(s):  
W A Hughes ◽  
R W Brownsey ◽  
R M Denton
Keyword(s):  
Pyruvate Dehydrogenase ◽  
Pyruvate Dehydrogenase Complex ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Subcellular Fractionation ◽  
Alpha Subunit ◽  
Fat Cells ◽  
Phosphorylated Proteins ◽  
Alpha Subunits ◽  
Dehydrogenase Complex

1. Intact rat epididymal fat-cells were incubated with 32Pi, and the intracellular proteins were separated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. One of the separated bands of phosphorylated proteins had an apparent subunit mol.wt. of 42 000, which is the same as that of the alpha-subunit of the pyruvate dehydrogenase complex. By using a combination of subcellular fractionation, immunoprecipitation with antiserum raised against pyruvate dehydrogenase complex and two-dimensional electrophoresis it was apparent that the incorporation into alpha-subunits accounted for 35–45% of the total incorporation into this band of phosphoproteins. 2. The increase in the initial activity of pyruvate dehydrogenase that follows brief exposure of fat-cells to insulin was shown to be associated with a decrease in the steady-state incorporation of 32P into the alpha-subunits of pyruvate dehydrogenase. 3. Tryptic peptide analysis of pyruvate dehydrogenase [32P]phosphate, labelled in intact fat-cells, indicated that three serine residues on the alpha-subunit were phosphorylated, corresponding to the three sites phosphorylated when purified pig heart pyruvate dehydrogenase was incubated with [gamma-32P]ATP. The relative phosphorylation of all three serine residues appeared to be similar in 32P-labelled alpha-subunits in both control and insulin-treated fat-cells.

scholarly journals Lymphocyte lineage-restricted tyrosine-phosphorylated proteins that bind PLC gamma 1 SH2 domains.

1992 ◽  
Vol 267 (19) ◽  
pp. 13610-13616 ◽  
Author(s):  
L.K. Gilliland ◽  
G.L. Schieven ◽  
N.A. Norris ◽  
S.B. Kanner ◽  
A Aruffo ◽  
...  
Keyword(s):  
Sh2 Domains ◽  
Phosphorylated Proteins

Systemic Analysis of Tyrosine Phosphorylated Proteins in Angiopoietin-1 Induced Signaling Pathway of Endothelial Cells

2007 ◽  
Vol 6 (8) ◽  
pp. 3278-3290 ◽  
Author(s):  
Young Mee Kim ◽  
Jawon Seo ◽  
Yung Hee Kim ◽  
Jaeho Jeong ◽  
Hye Joon Joo ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Signaling Pathway ◽  
Phosphorylated Proteins ◽  
Systemic Analysis ◽  
Angiopoietin 1

scholarly journals Identification and Specific Localization of Tyrosine-Phosphorylated Proteins in Trypanosoma brucei

2009 ◽  
Vol 8 (4) ◽  
pp. 617-626 ◽  
Author(s):  
Isabelle R. E. Nett ◽  
Lindsay Davidson ◽  
Douglas Lamont ◽  
Michael A. J. Ferguson
Keyword(s):  
Trypanosoma Brucei ◽  
Tyrosine Kinases ◽  
Immunofluorescence Microscopy ◽  
Significant Proportion ◽  
Tyrosine Residues ◽  
Phosphorylated Proteins ◽  
Cell Extracts ◽  
Proteomics Approach ◽  
Specific Localization ◽  
Multicellular Organisms

ABSTRACT Phosphorylation on tyrosine residues is a key signal transduction mechanism known to regulate intercellular and intracellular communication in multicellular organisms. Despite the lack of conventional tyrosine kinases in the genome of the single cell organism Trypanosoma brucei, phosphorylation on trypanosomal protein tyrosine residues has been reported for this parasite. However, the identities of most of the tyrosine-phosphorylated proteins and their precise site(s) of phosphorylation were unknown. Here, we have applied a phosphotyrosine-specific proteomics approach to identify 34 phosphotyrosine-containing proteins from whole-cell extracts of procyclic form T. brucei. A significant proportion of the phosphotyrosine-containing proteins identified in this study were protein kinases of the CMGC kinase group as well as some proteins of unknown function and proteins involved in energy metabolism, protein synthesis, and RNA metabolism. Interestingly, immunofluorescence microscopy using anti-phosphotyrosine antibodies suggests that there is a concentration of tyrosine-phosphorylated proteins associated with cytoskeletal structures (basal body and flagellum) and in the nucleolus of the parasite. This localization of tyrosine-phosphorylated proteins supports the idea that the function of signaling molecules is controlled by their precise location in T. brucei, a principle well known from higher eukaryotes.

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