基于iTRAQ技术的中医实热“上火”血清蛋白质组学特征分析

目的:筛选实热"上火"血清差异表达蛋白,为实热"上火"机制的深入研究提供科学依据。方法:采用相对和绝对定量同位素标记(iTRAQ)对血清蛋白质进行检测,结合生物信息学分析(GO、STRING)筛选实热"上火"与正常非"上火"人群差异显著的蛋白质。结果:与正常对照组比较,实热上火组血清筛选出49个差异蛋白质,其中上调的22个,下调的27个。载脂蛋白C3(Apo C3)、乳酸脱氢酶(LDH)和维生素K依赖蛋白S(PROS1)等显著上调,而载脂蛋白A4(Apo A4)、超氧化物歧化酶3(SOD3)和纤溶酶原激活物抑制剂1(PAI-1)等显著下调。结论:观察组脂质代谢、糖酵解异常与氧化应激、炎性反应的发生密切相关,且凝血功能下降,易造成出血,筛选的差异蛋白可为实热"上火"的生物学基础研究提供依据。     

Protein Z: A putative novel biomarker for early detection of ovarian cancer

Ovarian cancer (OC) has the highest mortality of all gynaecological cancers. Early diagnosis offers an approach to achieving better outcomes. We conducted a blinded‐evaluation of prospectively collected preclinical serum from participants in the multimodal group of the United Kingdom Collaborative Trial of Ovarian Cancer Screening. Using isobaric tags (iTRAQ) we identified 90 proteins differentially expressed between OC cases and controls. A second targeted mass spectrometry analysis of twenty of these candidates identified Protein Z as a potential early detection biomarker for OC. This was further validated by ELISA analysis in 482 serial serum samples, from 80 individuals, 49 OC cases and 31 controls, spanning up to 7 years prior to diagnosis. Protein Z was significantly down‐regulated up to 2 years pre‐diagnosis (p = 0.000000411) in 8 of 19 Type I patients whilst in 5 Type II individuals, it was significantly up‐regulated up to 4 years before diagnosis (p = 0.01). ROC curve analysis for CA‐125 and CA‐125 combined with Protein Z showed a statistically significant (p= 0.00033) increase in the AUC from 77 to 81% for Type I and a statistically significant (p= 0.00003) increase in the AUC from 76 to 82% for Type II. Protein Z is a novel independent early detection biomarker for Type I and Type II ovarian cancer; which can discriminate between both types. Protein Z also adds to CA‐125 and potentially the Risk of Ovarian Cancer algorithm in the detection of both subtypes.     

Effect of adipose‐derived stromal cells and BMP12 on intrasynovial tendon repair: A biomechanical,biochemical, and proteomics study

The outcomes of flexor tendon repair are highly variable. As recent efforts to improve healing have demonstrated promise for growth factor‐ and cell‐based therapies, the objective of the current study was to enhance repair via application of autologous adipose derived stromal cells (ASCs) and the tenogenic growth factor bone morphogenetic protein (BMP) 12. Controlled delivery of cells and growth factor was achieved in a clinically relevant canine model using a nanofiber/fibrin‐based scaffold. Control groups consisted of repair‐only (no scaffold) and acellular scaffold. Repairs were evaluated after 28 days of healing using biomechanical, biochemical, and proteomics analyses. Range of motion was reduced in the groups that received scaffolds compared to normal. There was no effect of ASC + BMP12 treatment for range of motion or tensile properties outcomes versus repair‐only. Biochemical assays demonstrated increased DNA, glycosaminoglycans, and crosslink concentration in all repair groups compared to normal, but no effect of ASC + BMP12. Total collagen was significantly decreased in the acellular scaffold group compared to normal and significantly increased in the ASC + BMP12 group compared to the acellular scaffold group. Proteomics analysis comparing healing tendons to uninjured tendons revealed significant increases in proteins associated with inflammation, stress response, and matrix degradation. Treatment with ASC + BMP12 amplified these unfavorable changes. In summary, the treatment approach used in this study induced a negative inflammatory reaction at the repair site leading to poor healing. Future approaches should consider cell and growth factor delivery methods that do not incite negative local reactions. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:630–640, 2016.     

The genome of Cyanothece 51142, a unicellular diazotrophic cyanobacterium important in the marine nitrogen cycle

Unicellular cyanobacteria have recently been recognized for their contributions to nitrogen fixation in marine environments, a function previously thought to be filled mainly by filamentous cyanobacteria such as Trichodesmium. To begin a systems level analysis of the physiology of the unicellular N₂-fixing microbes, we have sequenced to completion the genome of Cyanothece sp. ATCC 51142, the first such organism. Cyanothece 51142 performs oxygenic photosynthesis and nitrogen fixation, separating these two incompatible processes temporally within the same cell, while concomitantly accumulating metabolic products in inclusion bodies that are later mobilized as part of a robust diurnal cycle. The 5,460,377-bp Cyanothece 51142 genome has a unique arrangement of one large circular chromosome, four small plasmids, and one linear chromosome, the first report of a linear element in the genome of a photosynthetic bacterium. On the 429,701-bp linear chromosome is a cluster of genes for enzymes involved in pyruvate metabolism, suggesting an important role for the linear chromosome in fermentative processes. The annotation of the genome was significantly aided by simultaneous global proteomic studies of this organism. Compared with other nitrogen-fixing cyanobacteria, Cyanothece 51142 contains the largest intact contiguous cluster of nitrogen fixation-related genes. We discuss the implications of such an organization on the regulation of nitrogen fixation. The genome sequence provides important information regarding the ability of Cyanothece 51142 to accomplish metabolic compartmentalization and energy storage, as well as how a unicellular bacterium balances multiple, often incompatible, processes in a single cell.     

The inner of the two Muc2 mucin-dependent mucus layers in colon is devoid of bacteria

We normally live in symbiosis with ~10¹³ bacteria present in the colon. Among the several mechanisms maintaining the bacteria/host balance, there is limited understanding of the structure, function, and properties of intestinal mucus. We now demonstrate that the mouse colonic mucus consists of two layers extending 150 μm above the epithelial cells. Proteomics revealed that both of these layers have similar protein composition, with the large gel-forming mucin Muc2 as the major structural component. The inner layer is densely packed, firmly attached to the epithelium, and devoid of bacteria. In contrast, the outer layer is movable, has an expanded volume due to proteolytic cleavages of the Muc2 mucin, and is colonized by bacteria. Muc2^−/− mice have bacteria in direct contact with the epithelial cells and far down in the crypts, explaining the inflammation and cancer development observed in these animals. These findings show that the Muc2 mucin can build a mucus barrier that separates bacteria from the colon epithelia and suggest that defects in this mucus can cause colon inflammation.     

Dyggve-Melchior-Clausen syndrome: chondrodysplasia resulting from defects in intracellular vesicle traffic

Dyggve-Melchior-Clausen syndrome and Smith-McCort dysplasia are recessive spondyloepimetaphyseal dysplasias caused by loss-of-function mutations in dymeclin (Dym), a gene with previously unknown function. Here we report that Dym-deficient mice display defects in endochondral bone formation similar to that of Dyggve-Melchior-Clausen syndrome and Smith-McCort dysplasia, demonstrating functional conservation between the two species. Dym-mutant cells display multiple defects in vesicle traffic, as evidenced by enhanced dispersal of Golgi markers in interphase cells, delayed Golgi reassembly after brefeldin A treatment, delayed retrograde traffic of an endoplasmic reticulum-targeted Shiga toxin B subunit, and altered furin trafficking; and the Dym protein associates with multiple cellular proteins involved in vesicular traffic. These results establish dymeclin as a novel protein involved in Golgi organization and intracellular vesicle traffic and clarify the molecular basis for chondrodysplasia in mice and men.     

Retinal proteins modified by 4-hydroxynonenal: identification of molecular targets

The reactive aldehyde, 4-hydroxynonenal (HNE), is a product of lipid peroxidation that can covalently modify and inactivate proteins. Previously, we reported increased HNE modification of select retinal proteins resolved by one-dimensional gel electrophoresis in aged Fisher 344 x Brown Norway rats (Louie, J.L., Kapphahn, R.J., Ferrington, D.A., 2002. Proteasome function and protein oxidation in the aged retina. Exp. Eye Res. 75, 271-284). In the current study, quantitative assessment of HNE molar content using slot blot immunoassays showed HNE content is increased 30% in aged rat retina. In contrast, there was no age-related difference in HNE content in individual spots resolved by 2D gel electrophoresis suggesting the increased modification is likely on membrane proteins that are missing on 2D gels. The HNE-immunoreactive proteins resolved by 2D gel electrophoresis were identified by MALDI-TOF mass spectrometry. These proteins are involved in metabolism, chaperone function, and fatty acid transport. Proteins that were frequently modified and had the highest molar content of HNE included triosephosphate isomerase, alpha enolase, heat shock cognate 70 and betaB2 crystallin. Immunochemical detection of HNE adducts on retinal sections showed greater immune reaction in ganglion cells, photoreceptor inner segment, and the inner plexiform layer. Identification of HNE modified proteins in two alternative model systems, human retinal pigment epithelial cells in culture (ARPE19) and human donor eyes, indicated that triosephosphate isomerase and alpha enolase are generally modified. These results identify a common subset of proteins that contain HNE adducts and suggest that select retinal proteins are molecular targets for HNE modification.     

Proteomic analysis of differential protein expression in human atherosclerotic plaque progression

In this study, differential protein expression was assessed during human atherosclerotic plaque progression. A multifaceted approach was used in which differential protein expression was studied by two-dimensional (2D) gel electrophoresis and validated in individual patients using western blotting and immunohistochemistry. 2D profiles of whole-mount advanced stable lesions were compared to those of plaques containing a thrombus. Mass spectrometry analysis identified vinexin-beta and alpha1-antitrypsin (AAT) in the same spot that was differentially expressed in plaques with a thrombus. Immunohistochemistry and western blotting showed limited expression of both vinexin-beta and AAT in early lesions, whereas high expression of both proteins was found in advanced lesions. Differential expression of vinexin-beta in lesions with a thrombus compared to stable plaques could not be confirmed, indicating the importance of validation of proteomic analysis. For AAT, western blotting of 2D gels revealed expression of six isoforms in advanced plaques, one of which was confirmed to be solely expressed in thrombus-containing plaques. In conclusion, vinexin-beta is expressed in advanced human atherosclerotic plaques, but differential expression of this protein in lesions with a thrombus versus stable plaques could not be confirmed. However, this analysis revealed expression of six isoforms of AAT in advanced plaques, one of which was uniquely expressed in thrombus-containing plaques.     

Direct profiling of the cerebellum by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry: A methodological study in postnatal and adult mouse

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI MS) can detect substantial changes in expression of proteins in tissues, such as cancer cells. A more challenging problem is detecting the smaller changes expected in normal development or complex diseases. Here we address methodological issues regarding the acquisition and analysis of MALDI MS data from tissue sections, in a study of mouse cerebellum at different stages of development. Sections of the cerebellar cortex were analyzed at the peak of granule neuron production [postnatal day (P) 7], during synapse formation (P14), and in adults. Data were acquired (Voyager-DEtrade mark STR Biospectrometry Workstation; seven acquisitions of 50 shots per section, 3.5-50 kDa), preprocessed (Data Explorer 4.3), and averaged. Among 846 peaks detected, in at least 50% of at least one group, 122 showed significant group differences (Kruskal-Wallis ANOVA) after Bonferroni correction. Factor analyses revealed two age-related factors, possibly reflecting gradients of expression during development. Predictive analysis of microarrays generated a model from half of the sample that correctly predicted developmental groups for the second half. Intraclass correlation coefficients, measuring within-mouse consistency of peak heights from three tissue sections, were acceptable at lower m/z and for larger peaks at higher m/z. Low mass was the best predictor of significant group differences. The analysis demonstrates that MALDI MS of normal tissue sections at different ages can detect consistent, significant group differences. Further work is needed to increase the sensitivity of the methods and to apply them reliably to brain regions and to subproteomes with relevance to diverse brain functions and diseases.