ABSTRACT
The inverse relationship between HDL-C (high-density lipoprotein cholesterol) and cardiovascular disease is well established. However, it is consensus that the cholesterol content present in HDL does not capture its complexity, and other metrics need to be explored. HDL is a heterogeneous, protein-enriched particle with functions going beyond lipid metabolism. In this way, its protein content seems to be attractive to investigate its behavior in the face of pathologies. Many of the proteins with important function in HDL are in low abundance (<1% of total proteins), which makes their detection challenging. Quantitative proteomics allows detecting proteins with high precision and robustness in complex matrix. However, quantitative proteomics is still poorly explored in the context of HDL. In this sense, in the second chapter of this thesis, the analytical performance of two quantitative methodologies was carefully investigated. These methods achieved adequate linearity and high precision using labeled peptides in a pool HDL, in addition to comparable ability to differentiate proteins from HDL subclasses of healthy subjects. Another bottleneck that waits for a solution in proteomics is the lack of standardization in data processing and analysis after mass spectrometry acquisition. In addition, interest in the cardioprotective properties of omega-3 is growing, but little is known about its effects on the HDL proteome. Thus, in the third chapter of this thesis, we compared five protein quantification strategies using Skyline and MaxDIA software platforms in order to investigate the HDL proteome from mice submitted to a high-fat diet supplemented or not with omega-3. MaxDIA with label-free quantification (MaxLFQ) achieved high precision to show that polyunsaturated fatty acids remodel the HDL proteome to a less inflammatory profile. Therefore, the two studies presented in this thesis begin to open new paths for a deeper and more reliable understanding of HDL, both at the level of protein quantification by mass spectrometry and after data acquisition
A inversa relação entre HDL-C (do inglês, high-density lipoprotein cholesterol) e doenças cardiovasculares é bem estabelecida. No entanto, é consenso que o conteúdo de colesterol presente na HDL não captura sua complexidade, e outras métricas precisam ser exploradas. A HDL é uma partícula heterogênea, enriquecida em proteínas, com funções que vão além do metabolismo de lipídeos. Dessa forma, seu conteúdo proteico parece ser mais atrativo para exprimir seu comportamento frente às patologias. Muitas das proteínas com função importante estão em baixa abundância (<1% do total de proteínas), o que torna a detecção desafiadora. Métodos quantitativos de proteômica permitem detectar proteínas com alta precisão e robustez em matrizes complexas. No entanto, a proteômica quantitativa ainda é pouco explorada no contexto da HDL. Nesse sentido, no segundo capítulo dessa tese, a performance analítica de dois métodos quantitativos foi criteriosamente investigada, os quais alcançaram adequada linearidade e alta precisão usando peptídeos marcados em um pool de HDL, além de comparável habilidade em diferenciar as proteínas das subclasses da HDL de indivíduos saudáveis. Outro gargalo que aguarda por solução em proteômica é a falta de padronização no processamento e análise de dados após a aquisição por espectrometria de massas. Além disso, é crescente o interesse das propriedades cardioprotetivas do ômega-3, porém pouco se conhece sobre seus efeitos no proteoma da HDL. Então, no terceiro capítulo dessa tese, comparamos cinco estratégias de quantificação de proteínas utilizando os softwares Skyline e MaxDIA com o intuito de comparar o proteoma da HDL de camundongos submetidos a uma dieta hiperlipídica suplementados ou não com ômega-3. MaxDIA com quantificação label-free (MaxLFQ) apresentou alta precisão para mostrar que o ômega-3 remodela o proteoma da HDL para um perfil menos inflamatório. Portanto, os dois estudos apresentados nessa tesa começam a abrir novos caminhos para o entendimento mais profundo e confiável da HDL tanto por meio da quantificação das proteínas por espectrometria de massas quanto após à aquisição dos dados
Subject(s)
Proteomics/instrumentation , Hyperlipidemias/pathology , Cholesterol, HDL/analysis , Mass Spectrometry/methods , Cardiovascular Diseases/pathology , Diet/classification , Diet, High-Fat/adverse effectsABSTRACT
The local microenvironment is essential to stem cell-based therapy for ischemic stroke, and spatiotemporal changes of the microenvironment in the pathological process provide vital clues for understanding the therapeutic mechanisms. However, relevant studies on microenvironmental changes were mainly confined in the acute phase of stroke, and long-term changes remain unclear. This study aimed to investigate the microenvironmental changes in the subacute and chronic phases of ischemic stroke after stem cell transplantation. Herein, induced pluripotent stem cells (iPSCs) and neural stem cells (NSCs) were transplanted into the ischemic brain established by middle cerebral artery occlusion surgery. Positron emission tomography imaging and neurological tests were applied to evaluate the metabolic and neurofunctional alterations of rats transplanted with stem cells. Quantitative proteomics was employed to investigate the protein expression profiles in iPSCs-transplanted brain in the subacute and chronic phases of stroke. Compared with NSCs-transplanted rats, significantly increased glucose metabolism and neurofunctional scores were observed in iPSCs-transplanted rats. Subsequent proteomic data of iPSCs-transplanted rats identified a total of 39 differentially expressed proteins in the subacute and chronic phases, which are involved in various ischemic stroke-related biological processes, including neuronal survival, axonal remodeling, antioxidative stress, and mitochondrial function restoration. Taken together, our study indicated that iPSCs have a positive therapeutic effect in ischemic stroke and emphasized the wide-ranging microenvironmental changes in the subacute and chronic phases.
Subject(s)
Animals , Rats , Cell Differentiation , Disease Models, Animal , Ischemic Stroke , Proteomics , Stem Cell Transplantation/methods , Stroke/therapyABSTRACT
Resistance of tumor cells is a complex biological process involving multiple mechanisms and factors, in which anti-apoptosis is the most important cause of drug resistance. Previous studies have shown that the DNA binding activity of Runt related transcription factor 3 (RUNX3) increased prominently in Herceptin resistant gastric cancer cells (NCI N87R) while the relevance of which to drug resistance has not yet been confirmed. In this study, we employed CRISPR/Cas9 to establish RUNX3 knock-out cell line (△RUNX3/NCI N87R) to investigate the functions of RUNX3 in Herceptin resistance of NCI N87R cells and its potential mechanisms. We investigated proteomics profiling of △RUNX3/NCI N87R cells based on label free quantitative proteomics. Differentially expressed proteins were screened out according to fold change and significance level between △RUNX3/NCI N87R and NCI N87R cells. Pathway enrichment analysis was done using GeneAnalytics database, and gene ontology analysis was conducted by DAVID Bioinformatics Resources database. Protein-protein interaction networks were constructed based on STRING database. The results showed that △RUNX3/NCI N87R cells increased the sensitivity to Herceptin. Proteomic data demonstrated that the expression of 577 genes changed significantly in △RUNX3/NCI N87R cells, among which 191 genes were up-regulated while 386 ones down-regulated comparing with NCI N87R cells. Pathway analysis showed that autophagy, cell cycle, apoptosis, mitochondrial fatty acid β oxidation, neurogenic locus notch homolog protein 1 (NOTCH1), mammalian target of rapamycin (mTOR), Hedgehog and DNA damage response pathways exhibited notable changes based on pathway enrichment ratio and significance level (P < 0.05). These results indicated that RUNX3 knock-out altered multiple signaling pathways of NCI N87R cells. Western blotting manifested that the expression of autophagy regulatory molecules autophagy-related protein (ATG) 13, 7 and BECN1 increased remarkably while cell cycle molecules serine/threonine-protein kinase Chk2 (CHEK2) and apoptosis regulator Bcl-2 (BCL2) decreased prominently in △RUNX3/NCI N87R cells. The p-AKT expression decreased significantly in △RUNX3/NCI N87R cells compared with NCI N87R cells (P < 0.01) and was suppressed by Herceptin. These results indicated that RUNX3 knock-out altered cell cycle, increased inhibition to p-AKT by Herceptin, promoted autophagy and induced cell apoptosis of NCI N87R cells. These results suggested that RUNX3 may be a potential therapeutic target for reversing or reducing Herceptin resistance in gastric cancer cells.
ABSTRACT
In the past decade, relative proteomic quantification using isobaric labeling technology has developed into a key tool for comparing the expression of proteins in biological samples. Although its multiplexing capacity and flexibility make this a valuable technology for addressing various biological questions, its quantitative accuracy and precision still pose significant challenges to the reliability of its quantification results. Here, we give a detailed overview of the different kinds of isobaric mass tags and the advantages and disadvantages of the isobaric labeling method. We also discuss which precautions should be taken at each step of the isobaric labeling workflow, to obtain reliable quantification results in large-scale quantitative proteomics experiments. In the last section, we discuss the broad applications of the isobaric labeling technology in biological and clinical studies, with an emphasis on thermal proteome profiling and proteogenomics.
Subject(s)
Proteome/metabolism , Proteomics/methods , Reproducibility of Results , Tandem Mass Spectrometry/methodsABSTRACT
Objective To compare the difference of protein expression between the post-overwintering stage and the diapauses preparation stage in Culex pipiens pallens, so as to reveal the mechanisms underlying the overwintering diapause of Cx. pipienspallens. Methods A quantitative proteomic analysis was performed in Cx. pipiens pallens before and after overwintering diapause by using isobaric tags for relative and absolute quantification (iTRAQ) labeling. Results A total of 244 differentially expressed proteins were identified in Cx. pipiens pallens before and after overwintering diapause, including 126 up-regulated proteins and 118 down-regulated proteins. iTRAQ-based quantitative proteomic analysis revealed that these differentially expressed proteins were linked to function and energy production and conversion, lipid metabolism, remodeling of cytoskeleton, carbohydrate metabolism, protein transport, molecular chaperones, stress tolerance and metabolic enzymes. Conclusions This is the first study to identify the overwintering diapause-related proteins in Cx. pipiens pallens using proteomics tools, which reveals KEGG pathways and GO terms associated with the overwintering diapauses of Cx. pipiens pallens. Our findings provide additional understandings pertaining to the mechanisms underlying the overwintering diapauses of Cx. pipiens pallens.
ABSTRACT
Objective To compare the difference of protein expression between the post-overwintering stage and the diapauses preparation stage in Culex pipiens pallens, so as to reveal the mechanisms underlying the overwintering diapause of Cx. pipienspallens. Methods A quantitative proteomic analysis was performed in Cx. pipiens pallens before and after overwintering diapause by using isobaric tags for relative and absolute quantification (iTRAQ) labeling. Results A total of 244 differentially expressed proteins were identified in Cx. pipiens pallens before and after overwintering diapause, including 126 up-regulated proteins and 118 down-regulated proteins. iTRAQ-based quantitative proteomic analysis revealed that these differentially expressed proteins were linked to function and energy production and conversion, lipid metabolism, remodeling of cytoskeleton, carbohydrate metabolism, protein transport, molecular chaperones, stress tolerance and metabolic enzymes. Conclusions This is the first study to identify the overwintering diapause-related proteins in Cx. pipiens pallens using proteomics tools, which reveals KEGG pathways and GO terms associated with the overwintering diapauses of Cx. pipiens pallens. Our findings provide additional understandings pertaining to the mechanisms underlying the overwintering diapauses of Cx. pipiens pallens.
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Dendrobium denneanum have been used for a long time as rare medicinal herbs in traditional Chinese medicine. Our previous works found that ether extract of D. denneanum had higher anticancer activities than alcohol or water extract,thus with better development prospects. Quantitative proteomics based on SILAC technique was used to investigate the anticancer mechanism of D. denneanum on lung tumor cell line A549,and 4 855 proteins were detected in A549 cells. Quantitative proteomics experiments found that 193 proteins of A549 cells were up-regulated,and 44 proteins were down-regulated by ether extract of D. denneanum. Those proteins are associated with synthesis,transport and metabolism of biological macromolecules,chaperone,DNA repair,oxidoreductase,cell adhesion,cell cycle,apoptosis and autophagy. Through the function analysis of differentially expressed proteins,it was inferred that ether extract of D. denneanum caused cell protein metabolism disorder,endoplasmic reticulum stress response,abnormal self-repair mechanism of cells,damage of cell adhesion and proliferation; besides,it caused a dramatic increase in ROS level in A549 cells,and upset the balance of intracellular oxidation reduction system. Affected by the above factors,lung cancer cells initiated apoptosis and autophagy,which accelerated cell death. This research explains the anticancer mechanism of D. denneanum from the perspective of quantitative proteomics,and lays a foundation for future research and development of new anticancer drugs based on ether extract of D. denneanum.
Subject(s)
Animals , Humans , A549 Cells , Apoptosis , Dendrobium , Ether , Lung Neoplasms , ProteomicsABSTRACT
OBJECTIVE@#To analyze the effects of alterations in the expressions of methyltransferase on protein expression profiles in human nasopharyngeal carcinoma (NPC) cells and enrich the differential signaling pathways.@*METHODS@#The total protein was extracted from -knockout cell line CNE1 and the wild-type cell line CNE1, and the differentially expressed proteins were screened by tandem mass tag (TMT) labeled protein quantification technique and tandem mass spectrometry. GO analysis was used to annotate and enrich the differentially expressed proteins, and the KEGG database was used to enrich and analyze the pathways of the differential proteins.@*RESULTS@#With a fold change (FC)≥1.2 and < 0.05 as the screening standard, 2049 differentially expressed proteins were identified in CNE1 cells, among which 904 were up-regulated and 1145 were down-regulated. GO functional annotation results indicated that knockout caused characteristic changes in multiple biological processes (cell processes and regulation, cell movement, metabolic processes, and biosynthesis of cellular components), molecular functions (catalytic activity and molecular binding, transcription factor activity), and cellular components (cell membrane, organelle, macromolecular complex). KEGG analysis showed that the differentially expressed proteins were involved in an array of signaling pathways closely related to tumors, including MAPK, PI3K-Akt, Ras, Rap1, mTOR, Hippo, HIF-1, Wnt, AMPK, FoxO, ErbB, P53 and JAK-STAT.@*CONCLUSIONS@# knockout significantly changes the protein expression characteristics of NPC cells and affects a number of signal pathways closely related to tumors. The results provide evidence for investigation of the pathogenesis and therapeutic target screening of NPC.
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Chronic traumatic encephalopathy (CTE) is a distinct neurodegenerative disease that associated with repetitive head trauma. CTE is neuropathologically defined by the perivascular accumulation of abnormally phosphorylated tau protein in the depths of the sulci in the cerebral cortices. In advanced CTE, hyperphosphorylated tau protein deposits are found in widespread regions of brain, however the mechanisms of the progressive neurodegeneration in CTE are not fully understood. In order to identify which proteomic signatures are associated with CTE, we prepared RIPA-soluble fractions and performed quantitative proteomic analysis of postmortem brain tissue from individuals neuropathologically diagnosed with CTE. We found that axonal guidance signaling pathwayrelated proteins were most significantly decreased in CTE. Immunohistochemistry and Western blot analysis showed that axonal signaling pathway-related proteins were down regulated in neurons and oligodendrocytes and neuron-specific cytoskeletal proteins such as TUBB3 and CFL1 were reduced in the neuropils and cell body in CTE. Moreover, oligodendrocyte-specific proteins such as MAG and TUBB4 were decreased in the neuropils in both gray matter and white matter in CTE, which correlated with the degree of axonal injury and degeneration. Our findings indicate that deregulation of axonal guidance proteins in neurons and oligodendrocytes is associated with the neuropathology in CTE. Together, altered axonal guidance proteins may be potential pathological markers for CTE.
Subject(s)
Humans , Axons , Blotting, Western , Brain Injury, Chronic , Brain , Cell Body , Cerebral Cortex , Craniocerebral Trauma , Cytoskeletal Proteins , Gray Matter , Immunohistochemistry , Neurodegenerative Diseases , Neurons , Neuropathology , Neuropil , Oligodendroglia , tau Proteins , White MatterABSTRACT
Aim To explore the hypolipidemic mechanism of the total phenylpropanoid glycoside from Ligustrum robustum (Roxb. ) Blume (LRTPG) on hyperlipidemic hamsters using label-free quantitative proteomic technique. Methods The total protein was extracted from livers of model group and the group treated with LRTPG for label-free quantitative proteomics research. Results The proteomic data showed that a total of 2231 proteins were identified. And 549 proteins were found to be differentially expressed between model group and group treated with LRTPG. Among the 549 proteins, 93 proteins were up-regulated and 59 proteins were down-regulated, and 397 proteins had quantitative values only in model group or drug-administered group. Further, gene ontology (GO) analysis indicated that those differentially expressed proteins were primarily involved in an array of biological processes including metabolism, transport, oxidation-reduction, phosphorylation, signal transduction and lipid metabolism. KEGG pathway analysis revealed that these proteins were involved in several signal pathways including oxidative phosphorylation, non-alcoholic fatty liver dis-ease, PI3K-Akt, cAMP, and cGMP-PKG pathway. And some of these proteins were much related to the lipid metabolism, such as CD36, PK, HSS, GCK, ApoA I, Acly and FABP5. Conclusion The hypolipidemic effect of LRTPG may be related to CD36, PK, HSS, GCK, ApoA I, Acly and FABP5.
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Respiratory syncytial virus (RSV) is a leading cause of acute lower respiratory tract infections. Qingfei oral liquid (QFOL), a traditional Chinese medicine, is widely used in clinical treatment for RSV-induced pneumonia. The present study was designed to reveal the potential targets and mechanism of action for QFOL by exploring its influence on the host cellular network following RSV infection. We investigated the serum proteomic changes and potential biomarkers in an RSV-infected mouse pneumonia model treated with QFOL. Eighteen BALB/c mice were randomly divided into three groups: RSV pneumonia model group (M), QFOL-treated group (Q) and the control group (C). Serum proteomes were analyzed and compared using a label-free quantitative LC-MS/MS approach. A total of 172 protein groups, 1009 proteins, and 1073 unique peptides were successfully identified. 51 differentially expressed proteins (DEPs) were identified (15 DEPs when M/C and 43 DEPs when Q/M; 7 DEPs in common). Classification and interaction network showed that these proteins participated in various biological processes including immune response, blood coagulation, complement activation, and so forth. Particularly, fibrinopeptide B (FpB) and heparin cofactor II (HCII) were evaluated as important nodes in the interaction network, which was closely involved in coagulation and inflammation. Further, the FpB level was increased in Group M but decreased in Group Q, while the HCII level exhibited the opposite trend. These findings not only indicated FpB and HCII as potential biomarkers and targets of QFOL in the treatment of RSV pneumonia, but also suggested a regulatory role of QFOL in the RSV-induced disturbance of coagulation and inflammation-coagulation interactions.
Subject(s)
Animals , Biomarkers , Blood , Chromatography, Liquid , Disease Models, Animal , Drugs, Chinese Herbal , Pharmacology , Therapeutic Uses , Fibrinopeptide B , Genetics , Gene Expression Regulation , Heparin Cofactor II , Genetics , Lung , Pathology , Mice, Inbred BALB C , Proteome , Proteomics , Respiratory Syncytial Virus Infections , Blood , Drug Therapy , Respiratory Syncytial Viruses , Tandem Mass SpectrometryABSTRACT
OBJECTIVE Vascular dementia (VD) refers to a progressive decline in memory and cognitive function caused by chronic cerebral ischemia. 2-Vessels occlusion (2-VO) has been widely used as a model of VD. Xiao-Xu-Ming decoction, a well-known traditional Chinese medicine prescrip-tion,has been widely used to treat stroke and sequelae of stroke.The present study was to investigate the mechanism of Xiao-Xu-Ming decoction(XXM) against chronic cerebral ischemia injury in rats. METHODS After XXM treatment, rats were performed a memory testing with Morris water maze and motor ability testing using prehensile test and inclined screen test.Neuronal plasticity was observed by immunofluorescent staining with MAP2 antibody. Differentially expressed proteins of rat hippocampus were analyzed by Label-free quantitative proteomics. RESULTS XXM significantly alleviated 2-VO-induced learning and memory deficits, motor ability dysfunction, and neuronal plasticity injury in rats. The mechanism might be involved in up-regulation of 39 proteins and down-regulation of 13 proteins in the hippocampus of rats after XXM treatment vs 2-VO group rats.Gene ontology and pathway analysis showed that the regulated proteins are mainly involved in oxidation reduction process, intracellular signaling cascade process, and protein catabolic process, etc. The signal pathways are mainly involved in ubiquitin mediated proteolysis and phosphatidylinositol signaling system. CONCLUSION Current findings provide new insights into the molecular mechanisms of XXM on chronic cerebral ischemia.
ABSTRACT
Respiratory syncytial virus (RSV) is a leading cause of acute lower respiratory tract infections. Qingfei oral liquid (QFOL), a traditional Chinese medicine, is widely used in clinical treatment for RSV-induced pneumonia. The present study was designed to reveal the potential targets and mechanism of action for QFOL by exploring its influence on the host cellular network following RSV infection. We investigated the serum proteomic changes and potential biomarkers in an RSV-infected mouse pneumonia model treated with QFOL. Eighteen BALB/c mice were randomly divided into three groups: RSV pneumonia model group (M), QFOL-treated group (Q) and the control group (C). Serum proteomes were analyzed and compared using a label-free quantitative LC-MS/MS approach. A total of 172 protein groups, 1009 proteins, and 1073 unique peptides were successfully identified. 51 differentially expressed proteins (DEPs) were identified (15 DEPs when M/C and 43 DEPs when Q/M; 7 DEPs in common). Classification and interaction network showed that these proteins participated in various biological processes including immune response, blood coagulation, complement activation, and so forth. Particularly, fibrinopeptide B (FpB) and heparin cofactor II (HCII) were evaluated as important nodes in the interaction network, which was closely involved in coagulation and inflammation. Further, the FpB level was increased in Group M but decreased in Group Q, while the HCII level exhibited the opposite trend. These findings not only indicated FpB and HCII as potential biomarkers and targets of QFOL in the treatment of RSV pneumonia, but also suggested a regulatory role of QFOL in the RSV-induced disturbance of coagulation and inflammation-coagulation interactions.
Subject(s)
Animals , Biomarkers , Blood , Chromatography, Liquid , Disease Models, Animal , Drugs, Chinese Herbal , Pharmacology , Therapeutic Uses , Fibrinopeptide B , Genetics , Gene Expression Regulation , Heparin Cofactor II , Genetics , Lung , Pathology , Mice, Inbred BALB C , Proteome , Proteomics , Respiratory Syncytial Virus Infections , Blood , Drug Therapy , Respiratory Syncytial Viruses , Tandem Mass SpectrometryABSTRACT
In the present study, total membrane proteins from tumor cell lines including HepG2, Hep3B2, H226, Ovcar3 and N87 were extracted and digested with LysC and trypsin. The resulting peptide lysate were pre-fractionated and subjected to untargeted quantitative proteomics analysis using a high resolution mass spectrometer. The mass spectra were processed by the MaxQuant and the protein abundances were estimated using total peak area (TPA) method. A total of 6037 proteins were identified, and the analysis resulted in the identification of 2647 membrane proteins. Of those, tumor antigens and absorption, metabolism, disposition and elimination (ADME) proteins including UDP-glucuronosyltransferase, cytochrome P450, solute carriers and ATP-binding cassette transporters were detected and disclosed significant variations among the cell lines. The principal component analysis was performed for the cluster of cell lines. The results demonstrated that H226 is closely related with N87, while Hep3B2 aligned with HepG2. The protein cluster of Ovcar3 was apart from that of other cell lines investigated. By providing for the first time quantitative untargeted proteomics analysis, the results delineated the expression profiles of membrane proteins. These findings provided a useful resource for selecting targets of choice for anticancer therapy through advancing data obtained from preclinical tumor cell line models to clinical outcomes.
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Objective To discover the vital role of Rab27B in tumor cells and its potential molecular mechanism by means of quantitative proteomics analysis of Rab27B knockdown in MHCC97H.Methods The expression of Rab27B in MHCC97H cells was knocked down by the combination of Tet-on advanced inducible expression system and RNA interference technology.Then, proteins extracted from the cells were identified by LC-MS/MS system after FASP digestion and iTRAQ 4-plex labeling. Finally, the properties of differentially expressed proteins, including the subcellular localizations, biological processes and molecular functions, were analyzed by the bioinformatics method.Results There were 448 differentially expressed proteins (|Ratio|>1.21, P<0.05) identified in MHCC97H cells after Rab27B knock-down.The expression levels of 229 or 219 proteins were positively or negatively correlated with Rab27B, respectively. These differentially expressed proteins were mainly involved in vesicle transport, macromolecule localization, cellular response to stimulus.Furthermore, there were 26 differentially expressed proteins participating in 8 tumor-related signal pathways, eleven of which were in the focal adhesion signal pathway.Conclusion The analysis of quantitative proteomics in Rab27B-knockdown MHCC97H cell line by iTRAQ suggests that Rab27B not only has an impact on the exosomal secretion of tumor cells, but also regulates master proteins in signal pathways involved in cell proliferation and migration.
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Objective To identify the mediators of CDC42 signaling pathway involved in hepatitis B virus X protein (HBx)-mediated cellular transformation.Methods The mass defect-based pseudo-isobaric dimethyl labeling method (pIDL)was used to detect the differentially expressed proteins with a deficiency of CDC42.Furthermore,we conducted a gene ontology (GO)of differentially expressed proteins.Results and Conclusion We totally qualified 3409 proteins and found 220 differentially expressed proteins.Palladin,formin-like 1 (FMNL1)and keratin-19,which were implicated in cytoskeleton organization,were down-regulated with the deficiency of CDC42.Our results have provided candidate genes and proteins that may play an important role in HBx-mediated cellular transformation.
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By using the high resolution mass spectrometer TripleTOF 5600 , three kinds of standard proteins including bovine serum albumin ( BSA) , ovalbumin ( OVA) and lysozyme C( LYZC) were analyzed, and the correlationship between the ion intensity of mass spectrometry and the relative content of protein sample was investigated. The protein samples were digested by trypsion and diluted to 1-1024 fmol in 7 μL. The ion counts per second ( cps) were used to stand for the amounts of proteins and peptides. Then the correlation between sum of ion intensity ( cps) of all the peptides, number of peptides detected and the amount of proteins was investigated. By comparing the change of values of the same sample in three parallel experiments, a linear relationship between these indexes and the amount of proteins within 1-1024 fmol was found when the cps was more than 1000. Usually, the maximal ion intensity was no more than 1. 5 times of the minimum value for same peptide in triplicate experiments, which suggested that the 3 times or more change of ion intensity was the minimum threshold to determine the differences of proteins amounts in different samples. This study provides a relative quantitative analysis method using qualitative data of high resolution and high scan speed mass spectrometry, which can quickly and easily provide reference for biological and medical research.
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Objective To screen the glycoproteins as biomarkers for intracranial aneurysm (ⅠA) in cerebrospinal fluid (CSF) and evaluate the specificity and sensitivity of the biomarker candidates.Methods A complementary proteomic approach integrated with multidimensional chromatography was employed to simultaneously measure relative changes in the gylcoproteins of cerebrospinal fluid (CSF) obtained from patients with ruptured ⅠA (RIA) and unruptured ⅠA (UIA) compared to the healthy controls (HC) and disease controls (DC).One protein-receptor tyrosine kinase Axl with a unique change in RIA was validated in CSF and plasma.The sensitivity at 95％ specificity of Axl in CSF and plasma was evaluated with receiver operating characteristic curve (ROC curve).Results Firstly,a total of 294 glycoproteins were identified in human CSF with believable evidence.Secondly,the proteomic findings showed the quantitative changes in RIA and UIA as compared to HC and DC.Of 294 identified CSF proteins,59,24 and 33 proteins displayed quantitative changes unique to RIA,UIA or IA,respectively.At last,one of these unique proteins-receptor tyrosine kinase Axl with unique increase in RIA was confirmed both in CSF and plasma.ROC curve analysis showed that the sensitivity at 95％ specificity of Axl in CSF to differentiate RIA from UIA was 60％.When compared to CSF,the sensitivity at above setting in plasma to differentiate RIA from HC was 40％ and to differentiate RIA from UIA was 25％.Conclusions A glycoprotein biomarker Axl might be used as a promising biomarker to predict the rupture of ⅠA.The further investigation of the relations between Axl and IA formation as well as rupture might help to elucidate the underlying pathogenesis and find new therapeutic targets.
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Mass spectrometry is an important and powerful tool for protein quantification. With the in-depth development of quantitative proteomics, limitations of classic MS based quantification methods, such as complicated matrix interference and throughput/capacity limitation, start to appear. Recent progress of series novel MS based techniques provide effective solutions for the limitations of relative and absolute proteomic quantification, including synchronous precursor selection ( SPS ) , mass defect isobaric labeling, parallel reaction monitoring ( PRM) , multiplexing acquisition ( MSX) , and various novel data independent acquisition ( DIA) modes. Here we summarized the current limitations of quantitative proteomics, reviewed the latest MS based quantification approaches, and discussed the features and advantages of these novel techniques for quantitative proteomic application.
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Pesquisas em psiquiatria ainda necessitam de estudos não dirigidos por hipóteses para revelar fundamentos neurobiológicos e biomarcadores moleculares para distúrbios psiquiátricos. Metodologias proteômicas disponibilizam uma série de ferramentas para esses fins. Apresentamos o princípio de rotulação metabólica utilizando 15N para proteômica quantitativa e suas aplicações em modelos animais de fenótipos psiquiátricos com um foco particular em esquizofrenia. Exploramos o potencial de rotulação metabólica por 15N em diferentes tipos de experimentos, bem como suas considerações metodológicas
Psychiatric research is in need of non-hypothesis driven approaches to unravel the neurobiological underpinnings and identify molecular biomarkers for psychiatric disorders. Proteomics methodologies constitute a state-of-the-art toolbox for biomarker discovery in psychiatric research. Here we present the principle of in vivo 15N metabolic labeling for quantitative proteomics experiments and applications of this method in animal models of psychiatric phenotypes, with a particular focus on schizophrenia. Additionally we explore the potential of 15N metabolic labeling in different experimental set-ups as well as methodological considerations of 15N metabolic labeling-based quantification studies