Quantitative proteomic analysis of cold-responsive proteins in rice

Rice is susceptible to cold stress and with a future of climatic instability we will be unable to produce enough rice to satisfy increasing demand. A thorough understanding of the molecular responses to thermal stress is imperative for engineering cultivars, which have greater resistance to low temperature stress. In this study we investigated the proteomic response of rice seedlings to 48, 72 and 96?h of cold stress at 12-14°C. The use of both label-free and iTRAQ approaches in the analysis of global protein expression enabled us to assess the complementarity of the two techniques for use in plant proteomics. The approaches yielded a similar biological response to cold stress despite a disparity in proteins identified. The label-free approach identified 236 cold-responsive proteins compared to 85 in iTRAQ results, with only 24 proteins in common. Functional analysis revealed differential expression of proteins involved in transport, photosynthesis, generation of precursor metabolites and energy; and, more specifically, histones and vitamin B biosynthetic proteins were observed to be affected by cold stress.     

Monte carlo simulation-based algorithms for analysis of shotgun proteomic data

Two new statistical models based on Monte Carlo Simulation (MCS) have been developed to score peptide matches in shotgun proteomic data and incorporated in a database search program, MassMatrix (www.massmatrix.net). The first model evaluates peptide matches based on the total abundance of matched peaks in the experimental spectra. The second model evaluates amino acid residue tags within MS/MS spectra. The two models provide complementary scores for peptide matches that result in higher confidence in peptide identification when significant scores are returned from both models. The MCS-based models use a variance reduction technique that improves estimation precision. Due to the high computational expense of MCS-based models, peptide matches were prefiltered by other statistical models before further evaluation by the MCS-based models. Receiver operating characteristic analysis of the data sets confirmed that MCS-based models improved the overall performance of the MassMatrix search software, especially for low-mass accuracy data sets.     

Proteome analysis of male gametophyte development in rice anthers

We used proteomic analysis to investigate the changing patterns of protein synthesis during pollen development in anthers from rice plants grown under strictly controlled growth conditions. Cytological analysis and external growth measurements such as anther length, auricle distances and days before flowering were used to determine pollen developmental stages. This allowed the collection of synchronous anther materials representing six discrete pollen developmental stages. Proteins were extracted from the anther samples and separated by two-dimensional gel electrophoresis to produce proteome maps. The anther proteome maps of different developmental stages were compared and 150 protein spots, which were changed consistently during development, were analysed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry to produce peptide mass fingerprint (PMF) data. Database searches using these PMF data revealed the identities of 40 of the protein spots analyzed. These 40 proteins represent 33 unique gene products. Four protein spots that could not be identified by PMF analysis were analysed by N-terminal microsequencing. Multiple charge-isoforms of vacuolar acid invertase, fructokinase, beta-expansin and profilin were identified. These proteins are closely associated with sugar metabolism, cell elongation and cell expansion, all of which are cell activities that are essential to pollen germination. The existence of multiple isoforms of the same proteins suggests that during the process of pollen development some kind of post-translational modification of these proteins occurs.     

Quantitative proteomic analysis of Myc oncoprotein function

This study applies a new quantitative proteomics technology to the analysis of the function of the Myc oncoprotein in mammalian cells. Employing isotope-coded affinity tag (ICAT) reagent labeling and tandem mass spectrometry, the global pattern of protein expression in rat myc-null cells was compared with that of myc-plus cells (myc-null cells in which myc has been introduced) to generate a differential protein expression catalog. Expression differences among many functionally related proteins were identified, including reduction of proteases, induction of protein synthesis pathways and upregulation of anabolic enzymes in myc-plus cells, which are predicted to lead to increased cell mass (cell growth). In addition, reduction in the levels of adhesion molecules, actin network proteins and Rho pathway proteins were observed in myc-plus cells, leading to reduced focal adhesions and actin stress fibers as well as altered morphology. These effects are dependent on the highly conserved Myc Box II region. Our results reveal a novel cytoskeletal function for Myc and indicate the feasibility of quantitative whole-proteome analysis in mammalian cells.     

A proteomic analysis of leaf sheaths from rice

The proteins extracted from the leaf sheaths of rice seedlings were separated by 2-D PAGE, and analyzed by Edman sequencing and mass spectrometry, followed by database searching. Image analysis revealed 352 protein spots on 2-D PAGE after staining with Coomassie Brilliant Blue. The amino acid sequences of 44 of 84 proteins were determined; for 31 of these proteins, a clear function could be assigned, whereas for 12 proteins, no function could be assigned. Forty proteins did not yield amino acid sequence information, because they were N-terminally blocked, or the obtained sequences were too short and/or did not give unambiguous results. Fifty-nine proteins were analyzed by mass spectrometry; all of these proteins were identified by matching to the protein database. The amino acid sequences of 19 of 27 proteins analyzed by mass spectrometry were similar to the results of Edman sequencing. These results suggest that 2-D PAGE combined with Edman sequencing and mass spectrometry analysis can be effectively used to identify plant proteins.     

Implications of strain- and species-level sequence divergence for community and isolate shotgun proteomic analysis

The recent surge in microbial genomic sequencing, combined with the development of high-throughput liquid chromatography-mass-spectrometry-based (LC/LC-MS/MS) proteomics, has raised the question of the extent to which genomic information of one strain or environmental sample can be used to profile proteomes of related strains or samples. Even with decreasing sequencing costs, it remains impractical to obtain genomic sequence for every strain or sample analyzed. Here, we evaluate how shotgun proteomics is affected by amino acid divergence between the sample and the genomic database using a probability-based model and a random mutation simulation model constrained by experimental data. To assess the effects of nonrandom distribution of mutations, we also evaluated identification levels using in silico peptide data from sequenced isolates with average amino acid identities (AAI) varying between 76 and 98%. We compared the predictions to experimental protein identification levels for a sample that was evaluated using a database that included genomic information for the dominant organism and for a closely related variant (95% AAI). The range of models set the boundaries at which half of the proteins in a proteomic experiment can be identified to be 77-92% AAI between orthologs in the sample and database. Consistent with this prediction, experimental data indicated loss of half the identifiable proteins at 90% AAI. Additional analysis indicated a 6.4% reduction of the initial protein coverage per 1% amino acid divergence and total identification loss at 86% AAI. Consequently, shotgun proteomics is capable of cross-strain identifications but avoids most cross-species false positives.     

Quantitative proteomic analysis of the adipocyte plasma membrane

The adipocyte is a key regulator of mammalian metabolism. To advance our understanding of this important cell, we have used quantitative proteomics to define the protein composition of the adipocyte plasma membrane (PM) in the presence and absence of insulin. Using this approach, we have identified a high confidence list of 486 PM proteins, 52 of which potentially represent novel cell surface proteins, including a member of the adiponectin receptor family and an unusually high number of hydrolases with no known function. Several novel insulin-responsive proteins including the sodium/hydrogen exchanger, NHE6 and the collagens III and VI were also identified, and we provide evidence of PM-ER association suggestive of a unique functional association between these two organelles in the adipocyte. Together these studies provide a wealth of potential therapeutic targets for the manipulation of adipocyte function and a valuable resource for metabolic research and PM biology.     

Quantitative proteomic analysis of yeast DNA replication proteins

Chromatin is dynamically regulated, and proteomic analysis of its composition can provide important information about chromatin functional components. Many DNA replication proteins for example bind chromatin at specific times during the cell cycle. Proteomic investigation can also be used to characterize changes in chromatin composition in response to perturbations such as DNA damage, while useful information is obtained by testing the effects on chromatin composition of mutations in chromosome stability pathways. We have successfully used the method of stable isotope labeling by amino acids in cell culture (SILAC) for quantitative proteomic analysis of normal and pathological changes to yeast chromatin. Here we describe this proteomic method for analyzing changes to Saccharomyces cerevisiae chromatin, illustrating the procedure with an analysis of the changes that occur in chromatin composition as cells progress from a G1 phase block (induced by alpha factor) into S phase (in the presence of DNA replication inhibitor hydroxyurea).     

Quantitative proteomic analysis of drug-induced changes in mycobacteria

A new approach for qualitative and quantitative proteomic analysis using capillary liquid chromatography and mass spectrometry to study the protein expression response in mycobacteria following isoniazid treatment is discussed. In keeping with known effects on the fatty acid synthase II pathway, proteins encoded by the kas operon (AcpM, KasA, KasB, Accd6) were significantly overexpressed, as were those involved in iron metabolism and cell division suggesting a complex interplay of metabolic events leading to cell death.     

Comparative shotgun proteomic analysis of Clostridium acetobutylicum from butanol fermentation using glucose and xylose

Background

Cytokinin is a plant hormone that plays a crucial role in several processes of plant growth and development. In recent years, major breakthroughs have been achieved in the elucidation of the metabolism, the signal perception and transduction, as well as the biological functions of cytokinin. An important activity of cytokinin is the involvement in chloroplast development and function. Although this biological function has already been known for 50 years, the exact mechanisms remain elusive.

Results

To elucidate the effects of altered endogenous cytokinin content on the structure and function of the chloroplasts, chloroplast subfractions (stroma and thylakoids) from transgenic Pssu-ipt and 35S:CKX1 tobacco (Nicotiana tabacum) plants with, respectively, elevated and reduced endogenous cytokinin content were analysed using two different 2-DE approaches. Firstly, thykaloids were analysed by blue-native polyacrylamide gel electrophoresis followed by SDS-PAGE (BN/SDS-PAGE). Image analysis of the gel spot pattern thus obtained from thylakoids showed no substantial differences between wild-type and transgenic tobacco plants. Secondly, a quantitative DIGE analysis of CHAPS soluble proteins derived from chloroplast subfractions indicated significant gel spot abundance differences in the stroma fraction. Upon identification by MALDI-TOF/TOF mass spectrometry, these proteins could be assigned to the Calvin-Benson cycle and photoprotective mechanisms.

Conclusion

Taken together, presented proteomic data reveal that the constitutively altered cytokinin status of transgenic plants does not result in any qualitative changes in either stroma proteins or protein complexes of thylakoid membranes of fully developed chloroplasts, while few but significant quantitative differences are observed in stroma proteins.     

膜整合蛋白质的“鸟枪法”蛋白质组学分析策略

疾病状态下生物膜表面蛋白质分子标记的表达量和修饰状态会发生改变。但由于其低丰度和不易溶解等特性,制约了膜蛋白质组学的研究,同时也制约了相关药物靶标的设计。近年来,为克服这些困难,学者们提出了"鸟枪法"的膜蛋白质组学研究策略。基于此,本文论述了"鸟枪法"的蛋白质组学分析的基本过程及其后续的部分改进工作。随着新的策略不断被采用,更多膜蛋白质的拓扑学特征和功能的相关研究一定会走上新的台阶。     

Comparative proteomic analysis of indica and japonica rice varieties

Indica and japonica are two main subspecies of Asian cultivated rice (Oryza sativa L.) that differ clearly in morphological and agronomic traits, in physiological and biochemical characteristics and in their genomic structure. However, the proteins and genes responsible for these differences remain poorly characterized. In this study, proteomic tools, including two-dimensional electrophoresis and mass spectrometry, were used to globally identify proteins that differed between two sequenced rice varieties (93–11 and Nipponbare). In all, 47 proteins that differed significantly between 93–11 and Nipponbare were identified using mass spectrometry and database searches. Interestingly, seven proteins were expressed only in Nipponbare and one protein was expressed specifically in 93–11; these differences were confirmed by quantitative real-time PCR and proteomic analysis of other indica and japonica rice varieties. This is the first report to successfully demonstrate differences in the protein composition of indica and japonica rice varieties and to identify candidate proteins and genes for future investigation of their roles in the differentiation of indica and japonica rice.     

DBParser: web-based software for shotgun proteomic data analyses

We describe a web-based program called 'DBParser' for rapidly culling, merging, and comparing sequence search engine results from multiple LC-MS/MS peptide analyses. DBParser employs the principle of parsimony to consolidate redundant protein assignments and derive the most concise set of proteins consistent with all of the assigned peptide sequences observed in an experiment or series of experiments. The resulting reports summarize peptide and protein identifications from multidimensional experiments that may contain a single data set or combine data from a group of data sets, all related to a single analytical sample. Additionally, the results of multiple experiments, each of which may contain several data sets, can be compared in reports that identify features that are common or different. DBParser actively links to the primary mass spectral data and to public online databases such as NCBI, GO, and Swiss-Prot in order to structure contextually specific reports for biologists and biochemists.     

A proteomic analysis of cold stress responses in rice seedlings

Using proteomic analysis, an investigation aimed at a better understanding of the molecular adaptation mechanisms of cold stress was carried out in rice (Oryza sativa). The seedlings were exposed to a progressively low temperature stress treatment from normal temperature to 15, 10, and 5 degrees C. Proteins were extracted from the leaves collected from both control and stressed seedlings. By fractionation, approximately 1700 protein spots were separated and visualized on CBB-stained 2-D gels. Sixty protein spots were found to be up-regulated in responding to the progressively low temperature stress and displayed different dynamic patterns. As an initial work, 41 of these proteins were identified using MALDI-TOF MS or ESI/MS/MS. These cold responsive proteins, besides two proteins of unknown function, include four factors of protein biosynthesis, four molecular chaperones, two proteases, and eight enzymes involved in biosynthesis of cell wall components, seven antioxidative/detoxifying enzymes, and proteins linked to energy pathway, as well as a protein involved in signal transduction. The functional proteomes illuminate the facts, at least in plant cell, that protein quality control mediated by chaperones and proteases and enhancement of cell wall components play important roles in tolerance to cold stress. Using TargetP program, the subcellular localization of the identified proteins was analyzed. Proteins (43.9%) were predicted to be located in the chloroplasts, implying that chloroplast proteome is virtually subjective to cold stress. The physiological implications, revealed from the experimental data, are discussed in context of a complex metabolic network in plant cells responsive to cold stress.