蛋白质组学研究揭示的植物根盐胁迫响应机制

植物根是感知外界盐胁迫信号的首要器官。近年来,人们利用高通量的差异表达蛋白质组学技术,分析了水稻(Oryzasativa)、拟南芥(Arabidopsis thaliana)、大豆(Glycine max)、大麦(Hordeum vulgare)、小麦(Triticum aestivum)、木榄(Bruguieragymnorhiza)和匍匐翦股颖(Agrostis stolonifera)等植物根应答盐胁迫过程中蛋白质组的动态变化特征。通过整合植物根响应盐胁迫蛋白质组学研究结果,揭示了植物根部响应盐胁迫的多种调节机制,包括:利用多种信号通路与蛋白质磷酸化/去磷酸化感知并传递盐胁迫信号;通过膜蛋白与转运蛋白调节离子吸收/外排与区室化;通过抗氧化酶系统活性清除活性氧,并通过合成多种渗透调节物质与防御物质减轻细胞受到的伤害;通过改变参与糖类与能量代谢相关酶的表达调节能量代谢水平;通过细胞骨架动态重塑保持正常的细胞结构、物质运输与信息传递;通过转录、翻译与翻译后调控调节各种蛋白质的动态变化与相互作用;通过调控各种基础代谢与次生代谢水平保持细胞结构与代谢状态正常。     

Functional genetic and biophysical analyses of membrane disruption by human adenovirus

The identification of the adenovirus (AdV) protein that mediates endosome penetration during infection has remained elusive. Several lines of evidence from previous studies suggest that the membrane lytic factor of AdV is the internal capsid protein VI. While these earlier results imply a role for protein VI in endosome disruption, direct evidence during cell entry has not been demonstrated. To acquire more definitive proof, we engineered random mutations in a critical N-terminal amphipathic α-helix of VI in an attempt to generate AdV mutants that lack efficient membrane penetration and infection. Random mutagenesis within the context of the AdV genome was achieved via the development of a novel technique that incorporates both error-prone PCR and recombineering. Using this system, we identified a single mutation, L40Q, that significantly reduced infectivity and selectively impaired endosome penetration. Furthermore, we obtained biophysical data showing that the lack of efficient endosomalysis is associated with reduced insertion of the L40Q mutation in protein VI (VI-L40Q) into membranes. Our studies indicate that protein VI is the critical membrane lytic factor of AdV during cellular entry and reveal the biochemical basis for its membrane interactions.     

A morphometric filter improves the diagnostic value of morphometric analyses of frozen histopathological sections from mammary tumours.

Frozen sections of 202 consecutive breast tumour cases were analyzed by morphometric quantitation of nuclear features. Nuclei were selected at random. Conventional light microscope examination of the paraffin-embedded specimens revealed 144 cases of cancer and 56 benign tumours. Using multivariate discriminant analysis of morphometric features, all but two of the benign cases and 79% of the malignant tumours were correctly classified. When a morphometrically based dynamic filter set to exclude 'non-diagnostic' nuclei was used, the correctly classified malignant cases rose to 86% Morphometry is a fast, reproducible and efficient method that can be used in conjunction with the histomorphological diagnosis of mammary frozen sections. The combination of systematic sampling and an objective dynamic filter may be a powerful approach to quantitative analyses of tumours from other sites. However, it is also likely that efficiency can be improved by combining nuclear morphometric features with structural, histochemical and molecular biological data. The combination of traditional histomorphological examination with quantitative information may well increase the diagnostic accuracy in individual patients.     

ProteinSeq: high-performance proteomic analyses by proximity ligation and next generation sequencing

Despite intense interest, methods that provide enhanced sensitivity and specificity in parallel measurements of candidate protein biomarkers in numerous samples have been lacking. We present herein a multiplex proximity ligation assay with readout via realtime PCR or DNA sequencing (ProteinSeq). We demonstrate improved sensitivity over conventional sandwich assays for simultaneous analysis of sets of 35 proteins in 5 μl of blood plasma. Importantly, we observe a minimal tendency to increased background with multiplexing, compared to a sandwich assay, suggesting that higher levels of multiplexing are possible. We used ProteinSeq to analyze proteins in plasma samples from cardiovascular disease (CVD) patient cohorts and matched controls. Three proteins, namely P-selectin, Cystatin-B and Kallikrein-6, were identified as putative diagnostic biomarkers for CVD. The latter two have not been previously reported in the literature and their potential roles must be validated in larger patient cohorts. We conclude that ProteinSeq is promising for screening large numbers of proteins and samples while the technology can provide a much-needed platform for validation of diagnostic markers in biobank samples and in clinical use.     

Improved proteomic discovery by sample pre-fractionation using dual-column ion-exchange high performance liquid chromatography

Clinically relevant biomarkers are urgently needed for improving patient diagnosis, risk stratification, prognosis and therapeutic treatments. There is a particularly compelling motivation for identifying protein-based indicators of early-stage disease for more effective interventions. Despite recent progress, the proteomic discovery process remains a daunting challenge due to the sheer heterogeneity and skewed protein abundances in biofluids. Even the most advanced mass spectrometry systems exhibit limiting overall dynamic ranges and sensitivities relative to the needs of modern biomedical applications. To this end, we report the development of a robust, rapid, and reproducible high performance ion-exchange liquid chromatography pre-fractionation method that allows for improved proteomic detection coverage of complex biological specimens using basic tandem mass spectrometry screening procedures. This form of sample simplification prior to global proteomic profiling, which we refer to collectively as 'fractionomics', increases the number and diversity of proteins that can be confidently identified in tissue and cell lysates as compared to the straight analysis of unfractionated crude extracts.     

Rapid detection of microbial contamination in frozen vegetables by automated impedance measurements.

Automated impedance measurements can be used to rapidly assess whether a sample of frozen vegetables contains greater or less than 10(5) organisms per g. Microorganisms growing pureed food samples cause a change in the impedance of the medium when the organisms reach a threshold concentration of between 10(6) and 10(7) organisms per ml. Estimates of the concentration of microorganisms initially present in the food sample can be made by recording the time required for the organisms in the sample to replicate to threshold levels. In this study, the detection times for 357 samples of frozen vegetables were compared with standard plate counts for each sample. The agreement between the two methods in distinguishing samples containing more than 10(5) organisms per g was 92.6% for 257 assorted frozen vegetables and somewhat higher (93 to 96%) when separate cutoff times were used for each type of vegetable. The time required for analysis was about 5 h, compared to the 48 to 72 h required for standard plate counts.     

Rapid detection of microbial contamination in frozen vegetables by automated impedance measurements.

Automated impedance measurements can be used to rapidly assess whether a sample of frozen vegetables contains greater or less than 10(5) organisms per g. Microorganisms growing pureed food samples cause a change in the impedance of the medium when the organisms reach a threshold concentration of between 10(6) and 10(7) organisms per ml. Estimates of the concentration of microorganisms initially present in the food sample can be made by recording the time required for the organisms in the sample to replicate to threshold levels. In this study, the detection times for 357 samples of frozen vegetables were compared with standard plate counts for each sample. The agreement between the two methods in distinguishing samples containing more than 10(5) organisms per g was 92.6% for 257 assorted frozen vegetables and somewhat higher (93 to 96%) when separate cutoff times were used for each type of vegetable. The time required for analysis was about 5 h, compared to the 48 to 72 h required for standard plate counts.     

PAXgene fixation enables comprehensive metabolomic and proteomic analyses of tissue specimens by MALDI MSI

An alcohol-based non-crosslinking tissue fixative, PAXgene Tissue System, has been proposed as alternative fixation method to formalin, providing superior and morphological preservation. To date, metabolites have not been assessed in PAXgene-fixed tissues. The study focuses on a comparison between PAXgene and standard formalin fixation for metabolomic analysis by MALDI mass spectrometry imaging. Therefore, fifty-six samples from seven mice organs were fixed with PAXgene (PFPE) or formalin (FFPE), embedded in paraffin, and processed to a tissue microarray. PAXgene was able to spatially preserve metabolites in organs achieving an overlap of common metabolites ranging from 34 to 78% with FFPE. Highly similar signal intensities and visualization of molecules demonstrated negligible differences for metabolite imaging on PFPE compared to FFPE tissues. In addition, we performed proteomic analysis of intact proteins and peptides derived from enzymatic digestion. An overlap of 33 to 58% was found between FFPE and PFPE tissue samples in peptide analysis with a higher number of PFPE-specific peaks. Analysis of intact proteins achieved an overlap in the range of 0 to 28% owing to the poor detectability of cross-linked proteins in formalin-fixed tissues. Furthermore, metabolite and peptide profiles obtained from PFPE tissues were able to correctly classify organs independent of the fixation method, whereas a distinction of organs by protein profiles was only achieved by PAXgene fixation. Finally, we applied MALDI MSI to human biopsies by sequentially analyzing metabolites and peptides within the same tissue section. Concerning prospective studies, PAXgene can be used as an alternative fixative for multi-omic tissue analysis.     

Genomic and proteomic analyses of the agarolytic system expressed by Saccharophagus degradans 2-40

Saccharophagus degradans 2-40 (formerly Microbulbifer degradans 2-40) is a marine gamma-subgroup proteobacterium capable of degrading many complex polysaccharides, such as agar. While several agarolytic systems have been characterized biochemically, the genetics of agarolytic systems have been only partially determined. By use of genomic, proteomic, and genetic approaches, the components of the S. degradans 2-40 agarolytic system were identified. Five agarases were identified in the S. degradans 2-40 genome. Aga50A and Aga50D include GH50 domains. Aga86C and Aga86E contain GH86 domains, whereas Aga16B carries a GH16 domain. Novel family 6 carbohydrate binding modules (CBM6) were identified in Aga16B and Aga86E. Aga86C has an amino-terminal acylation site, suggesting that it is surface associated. Aga16B, Aga86C, and Aga86E were detected by mass spectrometry in agarolytic fractions obtained from culture filtrates of agar-grown cells. Deletion analysis revealed that aga50A and aga86E were essential for the metabolism of agarose. Aga16B was shown to endolytically degrade agarose to release neoagarotetraose, similarly to a beta-agarase I, whereas Aga86E was demonstrated to exolytically degrade agarose to form neoagarobiose. The agarolytic system of S. degradans 2-40 is thus predicted to be composed of a secreted endo-acting GH16-dependent depolymerase, a surface-associated GH50-dependent depolymerase, an exo-acting GH86-dependent agarase, and an alpha-neoagarobiose hydrolase to release galactose from agarose.     

Comparison of ultrafiltration units for proteomic and N-glycoproteomic analysis by the filter-aided sample preparation method

The precision with which the dissociation constant, K_D, can be obtained from isothermal titration calorimetry depends on, among other factors, the concentrations of the interacting species. The so-called c value—the ratio of analyte concentration to K_D—should fall in the range of 1 to 1000 for reliable K_D determination. On the basis of simulated, noise-free data, Biswas and Tsodikov [5] recently suggested an optimal c value of 5 to 20. By contrast, we find an optimum at c > 40 on determining the K_D confidence intervals through simulations containing noise levels typical of state-of-the-art microcalorimeters.     

Comparison of ultrafiltration units for proteomic and N-glycoproteomic analysis by the filter-aided sample preparation method

The filter-aided sample preparation (FASP) method allows gel-free processing of biological samples solubilized with detergents for proteomic analysis by mass spectrometry. In FASP detergents are removed by ultrafiltration, and after protein digestion peptides are separated from undigested material. Here we compare the effectiveness of different filtration devices for analysis of proteomes and glycoproteomes. We show that Microcon and Vivacon filtration units with nominal molecular weight cutoffs of 30,000 and 50,000 (30 and 50 k, respectively) are equally suitable for FASP, whereas Microcon 30 k units are most appropriate for mapping of N-glycosylation sites. The use of filters with these relatively large cutoffs facilitates depletion of detergents.     

Microfabricated modules for sample handling, sample concentration and flow mixing: application to protein analysis by tandem mass spectrometry

The comprehensive analysis of biological systems requires a combination of genomic and proteomic efforts. The large-scale application of current genomic technologies provides complete genomic DNA sequences, sequence tags for expressed genes (EST's), and quantitative profiles of expressed genes at the mRNA level. In contrast, protein analytical technology lacks the sensitivity and the sample throughput for the systematic analysis of all the proteins expressed by a tissue or cell. The sensitivity of protein analysis technology is primarily limited by the loss of analytes, due to adsorption to surfaces, and sample contamination during handling. Here we summarize our work on the development and use of microfabricated fluidic systems for the manipulation of minute amounts of peptides and delivery to an electrospray ionization tandem mass spectrometer. New data are also presented that further demonstrate the potential of these novel approaches. Specifically, we describe the use of microfabricated devices as modules to deliver femtomole amounts of protein digests to the mass spectrometer for protein identification. We also describe the use of a microfabricated module for the generation of solvent gradients at nl/min flow rates for gradient chromatography-tandem mass spectrometry. The use of microfabricated fluidic systems reduces the risk of sample contamination and sample loss due to adsorption to wetted surfaces. The ability to assemble dedicated modular systems and to operate them automatically makes the use of microfabricated systems attractive for the sensitive and large-scale analysis of proteins.     

Identification of genes required for Cf-dependent hypersensitive cell death by combined proteomic and RNA interfering analyses

Identification of hypersensitive cell death (HCD) regulators is essential to dissect the molecular mechanisms underlying plant disease resistance. In this study, combined proteomic and RNA interfering (RNAi) analyses were employed to identify genes required for the HCD conferred by the tomato resistance gene Cf-4 and the Cladosporium fulvum avirulence gene Avr4. Forty-nine proteins differentially expressed in the tomato seedlings mounting and those not mounting Cf-4/Avr4-dependent HCD were identified through proteomic analysis. Among them were a variety of defence-related proteins including a cysteine protease, Pip1, an operative target of another C. fulvum effector, Avr2. Additionally, glutathione-mediated antioxidation is a major response to Cf-4/Avr4-dependent HCD. Functional analysis through tobacco rattle virus-induced gene silencing and transient RNAi assays of the chosen 16 differentially expressed proteins revealed that seven genes, which encode Pip1 homologue NbPip1, a SIPK type MAP kinase Nbf4, an asparagine synthetase NbAsn, a trypsin inhibitor LeMir-like protein NbMir, a small GTP-binding protein, a late embryogenesis-like protein, and an ASR4-like protein, were required for Cf-4/Avr4-dependent HCD. Furthermore, the former four genes were essential for Cf-9/Avr9-dependent HCD; NbPip1, NbAsn, and NbMir, but not Nbf4, affected a nonadaptive bacterial pathogen Xanthomonas oryzae pv. oryzae-induced HCD in Nicotiana benthamiana. These data demonstrate that Pip1 and LeMir may play a general role in HCD and plant immunity and that the application of combined proteomic and RNA interfering analyses is an efficient strategy to identify genes required for HCD, disease resistance, and probably other biological processes in plants.