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Objective To analyze the construction of mouse liver phosphoproteome and phosphorylated kinases to provide useful information for integrating mouse kinase phosphorylation regulatory networks.Methods A new method was established to identify phosphoproteome from the mouse liver.First of all, liver protein was digested with trypsin before the resulting peptides were subjected to a two-step phosphopeptide enrichment and separation procedure consisting of TiO2 chro-maphy enrichment combined with high pHHPLC separation.Samples were injected onto aNanolC-Ultra-2Dplus system cou-pled to an AB-Sciex 5600 Triple TOF mass spectrometer instrument.Then data analysis was performed to provide information of new identified phosphorylation sites of kinase.Results and Conclusion Using our efficient and high-throughput platform, we reported the identification of 5386 phosophorylation sites and 4553 phosphopeptides from 1533 proteins of the mouse liver.126 new phosphorylation sites were identified from 116 kinases, which provides valuable infor-mation for phosphorylation networks in the mouse liver.
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Objective To investigate the role of protein phosphorylation in Pseudomonas aeruginosa (P.aeruginosa) strains in response to stress triggered by mouse macrophages.Methods The strong cation exchange-immobilized metal affinity chromatography (SCX-IMAC) was performed to enrich phosphopeptides.The nanoscale liquid chromatography coupled to tandem mass spectrometry (nano LC-MS/MS) was carried out to identify and analyze phosphoproteome.Results Fourteen phosphopeptides from twelve proteins were identified within thirty-one phosphorylation sites on serine,threonine and tyrosine residues.Fifty percent of these phosphorylated proteins were membrane proteins,indicating that their phosphorylation modification was more critical for bacteria in response to the stress.In terms of biological process of Gene Ontology,these identified proteins were involved in stress response,iron transport,anaerobic respiration,response to hydrogen peroxide and signal transduction by phosphorylation,etc.Conclusion These phosphorylated proteins in P.aeruginosa strains are necessary for signal transduction and their response to harsh environment within the macrophages,such as iron limitation,hypoxia and oxidative stress.This study provides evidence for further investigation on virulence and pathogenesis of P.aeruginosa.
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Objective To investigate the aberrant expression of phosphoproteome analysis of peripheral blood mononuclear cells( PBMCs) in patients with systemic lupus erythematosus( SLE) . Lay the foundation for further re-search of mechanism and treatment in patients with SLE. Methods Phosphopeptides were enriched using TiO2 from PBMCs of patients and healthy subjects, then analyzed by automated LC-MS/MS analysis. Phosphorylation sites were identified and quantitated by MASCOT and MaxQuant. Differential expressed proteins and peptides were screened based on the bioinformatics analysis. Results 1 035 phosphorylation sites were identified from SLE com-prared with normal subjects, 618 corresponding genes were screened out in annotation proteins. Pathway analyses showed 12 signaling pathways were identified. There were the most difference phosphorylation sites in mitogen acti-vated protein kinases( MAPK) signaling pathway. Conclusion Differently phosphorylated proteins and peptides can be detected in patients with SLE, which can be used as a mechanism of reference and supplement combined with metabolic pathway, and might be used as a potential target for treatment and research of SLE.
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Objective To fractionate phosphoproteome of mouse liver by two-dimensional (2D) liquid phase chromatography fractionation. Methods Phosphoproteins were extracted from lysates of normal mice livers by phosphate metal affinity chromatography (PMAC) resin. The phosphoproteins were exchanged by start buffer and separated by chromatofocusing in the first dimension. Then the fractions between pH 8.5 and pH 4.0 were separated by non-porous silica (NPS) reverse-phase high performance liquid chromatography (RP-HPLC). Finally, the UV maps were converted into gel-like maps by ProteoVue software. Results Phosphoproteins of mouse liver were successfully extracted and fractionated by two dimensional liquid phase chromatographic fractionation after concentration and desalt. Then pI/UV map of mouse liver phosphoproteome was successfully set-up. There are 16 fractions between pH 8.5 and pH 4.0 after chromatofocusing in the first dimension and the UV maps of each fraction were converted into pI/UV gel-like maps. Conclusions Combination of technique of phosphoproteins enrichment and 2-D liquid phase chromatographic fractionation is an effective approach to research phosphoproteome and the key base for further identification and investigation of phosphoproteins.
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Objective:To separate human kidney phosphoproteome by two-dimensional gel electrophoresis.Methods: The phosphorylated proteins from human kidney tissues were enriched with phosphate metal affinity chromatography(PMAC) resin.After being concentrated and desalted,the samples were separated by isoelectric focusing on first dimension and SDS electrophoresis on second dimension.Results: The phosphorylated proteins were successfully extracted from human kidney tissues and were separated by two-dimensional gel electrophoresis.Conclusion: Phosphoprotein enrichment technique combined with two-dimensional gel electrophoresis is an effective approach to study phosphoproteome,laying a foundation for further investigation of human kidney phosphoproteins.