Proteome Science最新文献

µ-Conotoxin GIIIB (µ-CTX GIIIB) is a polypeptide containing three disulfide bridges, produced by the sea snail Conus geographus. This study was aimed to explored the cytotoxic effects of µ-CTX GIIIB on mouse skeletal musculoblast (Sol8). Sol8 cells were exposed to ouabain and veratridine to establish the cell injury model, and then treated with µ-CTX GIIIB. CCK-8 was adopted to evaluate the cytotoxicity of µ-CTX GIIIB. Then, proteomics and transcriptome were conducted, and the explore the differentially expressed genes (DEGs) and differentially expressed proteins (DEPs) affected by µ-CTX GIIIB were found. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis was used to investigate the affected signaling pathways. µ-CTX GIIIB increased the cell survival rate of injured Sol8 cells. We found and identified 1,663 DEGs and 444 DEPs influenced by µ-CTX GIIIB. 106 pairs of correlated DEGs and DEPs were selected by combining transcriptome and proteome data. The results of KEGG and GO analysis showed that µ-CTX GIIB affected the cell cycle, apoptosis, DNA damage and repair, lipid metabolism and other biological processes of Sol8 cells. µ-CTX GIIIB could affected cell cycle regulation, DNA damage repair, and activation of tumor factors, with potential carcinogenic effects. Our results provide an important basis for the study of in vitro toxicity, the mechanism of toxicity and injury prevention by µ-CTX GIIIB.

The ability of the adult mammalian heart to regenerate can save the cardiac muscle from a loss of function caused by injury. Cardiomyocyte regeneration is a key aspect of research for the treatment of cardiovascular diseases. The mouse heart shows temporary regeneration in the first week after birth; thus, the newborn mouse heart is an ideal model to study heart muscle regeneration. In this study, proteomic analysis was used to investigate the differences in protein expression in the hearts of neonatal mice at days 1 (P1 group), 4 (P4 group), and 7 (P7 group). Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis showed changes in several groups of proteins, including the protein kinase A (PKA) signaling pathway. Moreover, it was found that PKA inhibitors and agonists regulated cardiomyocyte replication in neonatal mouse hearts. These findings suggest that PKA may be a target for the regulation of the cardiomyocyte cell cycle.

Background: Pseudomonas aeruginosa is well known for its intrinsic ability to resist a wide range of antibiotics, thus complicates treatment. Thus, understanding the response of the pathogen to antibiotics is important for developing new therapies. In this study, proteomic response of P. aeruginosa to the commonly used anti-pseudomonas antibiotics, ceftazidime (Caz) and meropenem (Mem) was investigated.

Methods: P. aeruginosa ATCC 9027, an antibiotic-susceptible strain, was exposed to sub-MIC values of antibiotics either Caz or Mem for 14 days to obtain E1 strains and then cultured in antibiotic-free environments for 10 days to obtain E2 strains. Proteomes of the initial and E1, E2 strains were identified and comparatively analyzed using isobaric tags for relative and absolute quantitation (iTRAQ) in cooperation with nano LC-MS/MS. Noted up and down-regulated proteins were confirmed with quantitative reverse transcriptase PCR (qRT-PCR).

Results: Overall, 1039 and 1041 proteins were identified in Caz and Mem-exposed strains, respectively. Upon antibiotic exposure, there were 7-10% up-regulated (Caz: 71, Mem: 85) and down-regulated (Caz: 106, Mem: 69) proteins (1.5-fold change cut-off). For both Caz and Mem, the DEPs were primarily the ones involved in metabolic process, membrane, virulence, protein synthesis, and antibiotic resistance in which proteins involved in antibiotics resistance tended to be up-regulated while proteins involved in protein synthesis and metabolic process were down-regulated. Noted proteins included beta-lactamase AmpC which was up-regulated and OprD which was down-regulated in both the antibiotic-exposed strains. Besides, biofilm formation related proteins TssC1 and Hcp1 in Caz- exposed strains and the membrane/ periplasmic proteins Azu and PagL in Mem-exposed strains were found significantly down-regulated. qRT-PCR results confirmed the expression change of AmpC, Hcp1 and OprD proteins.

Conclusion: Exposure of Pseudomonas aeruginosa to sub-MIC values of Caz and Mem resulted in around 10% change in its proteome. Not only proteins with confirmed roles in antibiotic resistance mechanisms changed their expression but also virulence- associated proteins. Both Caz and Mem response involved up-regulation of AmpC and down-regulation of OprD. While TssC1 and Hcp1 were responsible for Caz response, Azu and PagL were more likely involved in Mem response.

Background: Our previous work shows that increased matrix stiffness not only alters malignant characteristics of hepatocellular carcinoma (HCC) cells, but also attenuates metformin efficacy in treating HCC cells. Here, we identified differential membrane proteins related to matrix stiffness-mediated metformin resistance for better understand therapeutic resistance of metformin in HCC.

Methods: Differential membrane proteins in HCC cells grown on different stiffness substrates before and after metformin intervention were screened and identified using isobaric tags for relative and absolute quantification (iTRAQ) labeling coupled with the liquid chromatography-tandem mass spectrometry (LC-MS/MS), then bioinformatic analysis were applied to determine candidate membrane protein and their possible signaling pathway.

Results: A total of 5159 proteins were identified and 354 differential membrane proteins and membrane associated proteins, which might be associated with matrix stiffness-mediated metformin resistance were discovered. Then 94 candidate membrane proteins including 21 up-regulated protein molecules and 73 down-regulated protein molecules were further obtained. Some of them such as Annexin A2 (ANXA2), Filamin-A (FLNA), Moesin (MSN), Myosin-9 (MYH9), Elongation factor 2 (eEF2), and Tax1 binding Protein 3 (TAX1BP3) were selected for further validation. Their expressions were all downregulated in HCC cells grown on different stiffness substrates after metformin intervention. More importantly, the degree of decrease was obviously weakened on the higher stiffness substrate compared with that on the lower stiffness substrate, indicating that these candidate membrane proteins might contribute to matrix stiffness-mediated metformin resistance in HCC.

Conclusions: There was an obvious change in membrane proteins in matrix stiffness-mediated metformin resistance in HCC cells. Six candidate membrane proteins may reflect the response of HCC cells under high stiffness stimulation to metformin intervention, which deserve to be investigated in the future.

Background: DCM is a common cardiomyopathy worldwide, which is characterized by ventricular dilatation and systolic dysfunction. DCM is one of the most widespread diseases contributing to sudden death and heart failure. However, our understanding of its molecular mechanisms is limited because of its etiology and underlying mechanisms. Hence, this study explored the underlying molecular mechanism of dilated cardiomyopathy through integrative analysis of data mining, iTRAQ-PRM proteomics and bioinformatics METHODS: DCM target genes were downloaded from the public databases. Next, DCM was induced in 20 rats by 8 weeks doxorubicin treatment (2.5 mg/kg/week). We applied isobaric tags for a relative and absolute quantification (iTRAQ) coupled with proteomics approach to identify differentially expressed proteins (DEPs) in myocardial tissue. After association analysis of the DEPs and the key target genes, subsequent analyses, including functional annotation, pathway enrichment, validation, were performed.

Results: Nine hundred thirty-five genes were identified as key target genes from public databases. Meanwhile, a total of 782 DEPs, including 348 up-regulated and 434 down-regulated proteins, were identified in our animal experiment. The functional annotation of these DEPs revealed complicated molecular mechanisms including TCA cycle, Oxidative phosphorylation, Cardiac muscle contraction. Moreover, the DEPs were analyzed for association with the key target genes screened in the public dataset. We further determined the importance of these three pathways.

Conclusion: Our results demonstrate that TCA cycle, Oxidative phosphorylation, Cardiac muscle contraction played important roles in the detailed molecular mechanisms of DCM.

Objective: In this study, we aimed to identify differentially expressed heat shock protein (HSP) profiles in the villi and decidua from patients with early missed abortion (EMA).

Methods: By using high-throughput and high-precision parallel reaction monitoring (PRM)-based targeted proteomics techniques, this study examined the abundance of HSPs in the villi and decidua of 10 patients with EMA and 10 controls. Moreover, the abundance of 3 HSPs in the villi of another 22 patients with EMA and 22 controls was verified with Western blotting and immunohistochemistry (IHC).

Results: There were potential differences in the abundance of 16 HSPs and 42 polypeptides in human villi and decidua compared with those of the control group. Among them, HSP90AB1, HSPD1 and HSPA13 were downregulated in abundance in villi of patients with EMA, with a statistically significant difference, which was consistent with the verification results of Western blots and IHC.

Conclusion: Using a PRM-based targeted proteomics technique, this study is the first to screen and quantitatively analyze the expression profile of HSPs in the villi and decidua of patients with EMA. The significant downregulation of HSP90AB1, HSPD1 and HSPA13 was found to have a potentially intimate association with the occurrence of EMA. The findings in our study may provide novel potential research targets related to HSPs for the pathogenesis, prevention and treatment of EMA.

Background: Effective diagnostic biomarkers for aortic aneurysm (AA) that are detectable in blood tests are required because early detection and rupture risk assessment of AA can provide insights into medical therapy and preventive treatments. However, known biomarkers for AA lack specificity and reliability for clinical diagnosis.

Methods: We performed proteome analysis of serum samples from patients with atherosclerotic thoracic AA (TAA) and healthy control (HC) subjects to identify diagnostic biomarkers for AA. Serum samples were separated into low-density lipoprotein, high-density lipoprotein, and protein fractions, and the major proteins were depleted. From the proteins identified in the three fractions, we narrowed down biomarker candidates to proteins uniformly altered in all fractions between patients with TAA and HC subjects and evaluated their capability to discriminate patients with TAA and those with abdominal AA (AAA) from HC subjects using receiver operating characteristic (ROC) analysis. For the clinical validation, serum concentrations of biomarker candidates were measured in patients with TAA and AAA registered in the biobank of the same institute, and their capability for the diagnosis was evaluated.

Results: Profilin 1 (PFN1) and complement factor D (CFD) showed the most contrasting profiles in all three fractions between patients with TAA and HC subjects and were selected as biomarker candidates. The PFN1 concentration decreased, whereas the CFD concentration increased in the sera of patients with TAA and AAA when compared with those of HC subjects. The ROC analysis showed that these proteins could discriminate patients with TAA and AAA from HC subjects. In the validation study, these candidates showed significant concentration differences between patients with TAA or AAA and controls. PFN1 and CFD showed sufficient area under the curve (AUC) in the ROC analysis, and their combination further increased the AUC. The serum concentrations of PFN1 and CFD also showed significant differences between patients with aortic dissection and controls in the validation study.

Conclusion: PFN1 and CFD are potential diagnostic biomarkers for TAA and AAA and measurable in blood samples; their diagnostic performance can be augmented by their combination. These biomarkers may facilitate the development of diagnostic systems to identify patients with AA.

Background: Subarachnoid haemorrhage (SAH) is one of the most severe forms of stroke in which delayed cerebral ischemia is one of the major complications. Neurointensive care aims at preventing and treating such complications and identification of biomarkers of early signs of ischemia might therefore be helpful.

Methods: We aimed at describing proteome profile in cerebral microdialysate in four patients with aneurysmal SAH using two dimensional gel electrophoresis in combination with mass spectrometry in search for new biomarkers for delayed cerebral ischemia and to investigate if there were temporal fluctuations in those biomarkers over time after aneurysmal bleed.

Results: The results showed transthyretin in nine different proteoforms (1001, 1102, 2101, 3101, 4101, 4102, 5001, 5101, 6101) in cerebral microdialysate samples from four patients having sustained SAH. Several proteoforms show highly differing levels and pooled analysis of all samples showed varying optical density related to time from aneurysmal bleed, indicating a temporal evolution.

Conclusions: Transthyretin proteoforms have not earlier been shown in cerebral microdialysate after SAH and we describe differing levels based on proteoform as well as time from subarachnoid bleed. Transthyretin is well known to be synthetized in choroid plexus, whilst intraparenchymal synthesis remains controversial. The results need to be confirmed in larger studies in order to further describe transthyretin.

Chronic hepatitis B is a significant public health problem and complex pathologic process, and unraveling the underlying mechanisms and pathophysiology is of great significance. Data independent acquisition mass spectrometry (DIA-MS) is a label-free quantitative proteomics method that has been successfully applied to the study of a wide range of diseases. The aim of this study was to apply DIA-MS for proteomic analysis of patients with chronic hepatitis B. We performed comprehensive proteomics analysis of protein expression in serum samples from HBV patients and healthy controls by using DIA-MS. Gene Ontology (GO) terms, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, and protein network analysis were performed on differentially expressed proteins and were further combined with literature analysis. We successfully identified a total of 3786 serum proteins with a high quantitative performance from serum samples in this study. We identified 310 differentially expressed proteins (DEPs) (fold change > 1.5 and P value < 0.05 as the criteria for a significant difference) between HBV and healthy samples. A total of 242 upregulated proteins and 68 downregulated proteins were among the DEPs. Some protein expression levels were significantly elevated or decreased in patients with chronic hepatitis B, indicating a relation to chronic liver disease, which should be further investigated.

Background: Lumbrokinase derived from earthworms, Lumbricus rubellus is known to have fibrinolytic enzymes that have potential as therapeutic drugs due to its ability to dissolve fibrin. The current study is aimed to purify the Lumbrokinase from L. rubellus and identify its protein component.

Methods: Water extract of local earthworm Lumbricus rubellus revealed several proteins. Therefore, to identify its protein component, purification through HiPrep DEAE fast flow and proteomic analysis were conducted prior to identifications. A combination of two-dimension gel electrophoresis (2DE) and electrospray ionization mass spectrometry analysis was used to identify the purified fractions.

Results: The purified fractions contain five protein bands, namely F25-1, F25-2, F85-1, F85-2, and F85-3, which displayed strong fibrinogenolytic activity. F25 fractions showed fibrinogenolytic activity of 974.85 U/mg, while F85 fractions showed higher activity of 1,484.11 U/mg. Fractions F85-1, F85-2, and F85-3 showed molecular weights of 42.6 kDa, 27.03 kDa, and 14 kDa, respectively and were identified as Lumbrokinase iso-enzymes.

Conclusion: This preliminary study indicates that the F25 and F85 fractions are similar to published fibrinolytic protease-1 and lumbrokinase, respectively, in terms of their amino acid sequence.