Shotgun proteomic analysis of human-induced sputum

Induced sputum is a readily accessible biological fluid whose composition may alter as a consequence of disease. To date, however, the proteins that routinely populate this biofluid are largely unknown, in part due to the technical difficulties in processing such mucin-rich samples. To provide a catalogue of sputum proteins, we have surveyed the proteome of human-induced sputum (sputome). A combination of 2-D gel analysis and GeLC-MS/MS allowed a total of 191 human proteins to be confidently assigned. In addition to the expected components, several hitherto unreported proteins were found to be present, including three members of the annexin family, kallikreins 1 and 11, and peroxiredoxins 1, 2 and 5. Other sets of proteins identified included four proteins previously annotated as hypothetical or conserved hypothetical. Taken together, these data represent the first extensive survey of the proteome of induced sputum and provide a platform for future identification of biomarkers of lung disease.     

Shotgun proteomic analysis of Chlamydia trachomatis

Chlamydiae are widespread bacterial pathogens responsible for a broad range of diseases, including sexually transmitted infections, pneumonia and trachoma. To validate the existence of hitherto hypothetical proteins predicted from recent chlamydial genome sequencing projects and to examine the patterns of expression of key components at the protein level, we have surveyed the expressed proteome of Chlamydia trachomatis strain L2. A combination of two-dimensional gel analysis, multi-dimensional protein identification (MudPIT) and nanocapillary liquid chromatography-tandem mass spectrometry allowed a total of 328 chlamydial proteins to be unambiguously assigned. Proteins identified as being expressed in the metabolically inert form, elementary body, of Chlamydia include the entire set of predicted glycolytic enzymes, indicating that metabolite flux rather than de novo synthesis of this pathway is triggered upon infection of host cells. An enzyme central to cell wall biosynthesis was also detected in the intracellular form, reticulate body, of Chlamydia, suggesting that the peptidoglycan is produced during growth within host cells. Other sets of proteins identified include 17 outer membrane-associated proteins of potential significance in vaccine studies and 67 proteins previously annotated as hypothetical or conserved hypothetical. Taken together, >/=35% of the predicted proteome for C. trachomatis has been experimentally verified, representing the most extensive survey of any chlamydial proteome to date.     

Comparative proteomic analysis provides new insights into mulberry dwarf responses in mulberry (Morus alba L.)

Mulberry dwarf (MD) is a serious infectious disease of mulberry caused by phytoplasma. Infection with MD phytoplasma results in stress phenotypes of yellowing, phyllody, stunting, proliferation, and witches' broom. Physiological and biochemical analysis has shown that infection with MD phytoplasma causes an increase in soluble carbohydrate and starch content, and a decrease in the net photosynthesis rate, carboxylation efficiency, and pigment content of leaves. Furthermore, damage to the chloroplast ultrastructure was detected in infected leaves. To better understand the pathogen‐stress response of mulberry (Morus alba L.) to MD phytoplasma, we conducted a comparative proteomic analysis using 2‐DE of infected and healthy leaves. Among 500 protein spots that were reproducibly detected, 20 were down‐regulated and 17 were up‐regulated. MS identified 16 differentially expressed proteins. The photosynthetic proteins rubisco large subunit, rubisco activase, and sedoheptulose‐1,7‐bisphosphatase showed enhanced degradation in infected leaves. Based these results, a model for the occurrence mechanism of MD is proposed. In conclusion, this study provides new insights into the mulberry response to MD phytoplasma infection.     

Shotgun proteomic analysis of the unicellular alga Ostreococcus tauri

Ostreococcus tauri is a unicellular green alga and amongst the smallest and simplest free-living eukaryotes. The O. tauri genome sequence was determined in 2006. Molecular, physiological and taxonomic data that has been generated since then highlight its potential as a simple model species for algae and plants. However, its proteome remains largely unexplored. This paper describes the global proteomic study of O. tauri, using mass spectrometry-based approaches: phosphopeptide enrichment, cellular fractionation, label-free quantification and (15)N metabolic labeling. The O. tauri proteome was analyzed under the following conditions: sampling at different times during the circadian cycle, after 24h of illumination, after 24h of darkness and under various nitrogen source supply levels. Cell cycle related proteins such as dynamin and kinesin were significantly up-regulated during the daylight-to-darkness transition. This is reflected by their higher intensity at ZT13 and this transition phase coincides with the end of mitosis. Proteins involved in several metabolic mechanisms were found to be up-regulated under low nitrogen conditions, including carbon storage pathways, glycolysis, phosphate transport, and the synthesis of inorganic polyphosphates. Ostreococcus tauri responds to low nitrogen conditions by reducing its nitrogen assimilation machinery which suggests an atypical adaptation mechanism for coping with a nutrient-limited environment.     

Shotgun proteomic analysis of long-distance drought signaling in rice roots

Rice (Oryza sativa L. cv. IR64) was grown in split-root systems to analyze long-distance drought signaling within root systems. This in turn underpins how root systems in heterogeneous soils adapt to drought. The approach was to compare four root tissues: (1) fully watered; (2) fully droughted and split-root systems where (3) one-half was watered and (4) the other half was droughted. This was specifically aimed at identifying how droughted root tissues altered the proteome of adjacent wet roots by hormone signals and how wet roots reciprocally affected dry roots hydraulically. Quantitative label-free shotgun proteomic analysis of four different root tissues resulted in identification of 1487 nonredundant proteins, with nearly 900 proteins present in triplicate in each treatment. Drought caused surprising changes in expression, most notably in partially droughted roots where 38% of proteins were altered in level compared to adjacent watered roots. Specific functional groups changed consistently in drought. Pathogenesis-related proteins were generally up-regulated in response to drought and heat-shock proteins were totally absent in roots of fully watered plants. Proteins involved in transport and oxidation-reduction reactions were also highly dependent upon drought signals, with the former largely absent in roots receiving a drought signal while oxidation-reduction proteins were strongly present during drought. Finally, two functionally contrasting protein families were compared to validate our approach, showing that nine tubulins were strongly reduced in droughted roots while six chitinases were up-regulated, even when the signal arrived remotely from adjacent droughted roots.     

Shotgun proteomic analysis of sarcoplasmic reticulum preparations from rabbit skeletal muscle

To obtain a comprehensive understanding of proteins involved in excitation–contraction coupling, a catalog of proteins from sarcoplasmic reticulum (SR) membrane fractions of New Zealand white rabbit skeletal muscle was analyzed by an optimized shotgun proteomic method. Light and heavy SR membrane fractions were obtained by nonlinear sucrose gradient centrifugation and separated by 1DE followed by a highly reproducible, automated LC‐MS/MS on the hybrid linear ion trap (LTQ) Orbitrap mass spectrometer. By integrating as low as 1% false discovery rate as one of the features for quality control method, 483 proteins were identified from both of the two independent SR preparations. Proteins involved in calcium release unit complex, including ryanodine receptor 1, dihydropyridine receptor, calmodulin, triadin, junctin, and calsequestrin, were all detected, which offered validation for this protein identification method. Rigorous bioinformatics analysis was performed. Protein pI value, molecular weight range, hydrophobicity index, and transmembrane region were calculated using bioinformatics softwares. Eighty‐three proteins were classified as hydrophobic proteins and 175 proteins were recognized as membrane proteins. Based on the proteomic analysis results, we found as the first time that not only transverse tubule but also mitochondrion physically connected to SR. The complete mapping of these proteomes may help in the elucidation of the process of excitation–contraction coupling and excitation–metabolism coupling.     

Shotgun proteomic analysis of soybean embryonic axes during germination under salt stress

Seed imbibition and radicle emergence are generally less affected by salinity in soybean than in other crop plants. In order to unveil the mechanisms underlying this remarkable salt tolerance of soybean at seed germination, a comparative label‐free shotgun proteomic analysis of embryonic axes exposed to salinity during germination sensu stricto (GSS) was conducted. The results revealed that the application of 100 and 200 mmol/L NaCl stress was accompanied by significant changes (>2‐fold, P<0.05) of 97 and 75 proteins, respectively. Most of these salt‐responsive proteins (70%) were classified into three major functional categories: disease/defense response, protein destination and storage and primary metabolism. The involvement of these proteins in salt tolerance of soybean was discussed, and some of them were suggested to be potential salt‐tolerant proteins. Furthermore, our results suggest that the cross‐protection against aldehydes, oxidative as well as osmotic stress, is the major adaptive response to salinity in soybean.     

Shotgun proteomic analysis of cerebrospinal fluid using off-gel electrophoresis as the first-dimension separation

Shotgun proteomic analysis usually employs multidimensional separations with the first dimension most commonly being strong cation exchange (SCX) liquid chromatography (LC). SCX-LC is necessarily a serial process for preparation of multiple samples. Here, we apply a newly available tool, off-gel electrophoresis (OGE), for first-dimension separation of peptide mixtures from digests of cerebrospinal fluid (CSF), a complex and low total protein-containing sample. OGE first-dimension fractionation enabled identification of a total of 156 unique proteins compared to 115 identified in previous work using first-dimension SCX fractionation. OGE can be used to process multiple samples unattended with easy retrieval of the separated fractions. Thus, shotgun analysis using OGE as the first-dimension separation offers a significant advantage both in terms of sample throughput as well as increased numbers of identified proteins.     

Shotgun proteomic analysis of a chromatophore-enriched preparation from the purple phototrophic bacterium Rhodopseudomonas palustris

A proteomics approach was evaluated for analysis of photosyntheis-related proteins that are characteristic of chromatophores, particles derived from purple phototrophic bacterial intracytoplasmic membranes. Proteins of purified chromatophores from Rhodopseudomonas palustris were solubilized and digested with trypsin, to create a collection of peptides that were fractionated by liquid chromatography. Peptide sequences were determined and assigned to specific proteins by analysis of tandem mass spectra of peptides, and comparison to a library derived from the recently determined R. palustris genome sequence. A total of 300 proteins were detected with a probability value >/=0.9, and the number of proteins detected increased to 345 when the minimum probability value was reduced to 0.5. Membrane-integral proteins of the reaction center, cytochrome b/c (1), light-harvesting and ATPase complexes were used as controls to assess how well this approach performs with hydrophobic proteins. New genes were identified, and tentatively designated as encoding photosynthesis-related proteins. We conclude that this approach is a powerful method to evaluate the possible existence of new photosynthesis-related proteins (and genes), although alternative methods are needed to evaluate the exact functions of newly discovered genes.     

Shotgun proteomic analysis for detecting differentially expressed proteins in the reduced culm number rice

To survey protein expression patterns in the reduced culm number (RCN) rice, a comparative shotgun proteomic analysis was conducted. For large-scale protein identification, multidimensional protein identification technology (MudPIT) coupled with pre-fractionation of plant shoot proteins led to the identification of 3004 non-redundant rice proteins. By statistically comparing relative amounts of 1353 reproducibly identified proteins between the RCN rice and the wild-type rice, 44 differentially expressed proteins were detected, where 42 proteins were increased and 2 proteins were decreased in the RCN rice. These proteins appear to have roles in glycolysis, trichloroacetic acid cycle, secondary metabolism, nutrient recycling, and nucleotide metabolism and repair. Consequently, we hypothesized that the RCN rice might fail to maintain sugar nutrient homeostasis. This was confirmed with the observation that the sucrose concentration was increased significantly in the RCN rice compared with the wild-type rice. Also, the RCN rice showed a hypersensitive response to exogenous sucrose treatment.     

小麦蓝矮植原体染色体DNA的分离

[目的]分离小麦蓝矮(WBD)植原体染色体DNA,并建立WBD植原体染色体分离纯化体系.[方法]采用差速离心和脉冲电泳(PFGE)方法富集纯化WBD植原体染色体DNA,并通过PCR和Southern blot进行检测验证,实时荧光定量PCR方法对分离纯化效果进行定量检测.[结果]脉冲电泳凝胶中出现一条大小约为650 kb的条带,经PCR检测和Southern blot分析表明该条带为WBD植原体的染色体DNA.实时荧光定量PCR检测结果表明采用差速离心与脉冲电泳结合的方法可以将WBD植原体基因组的相对拷贝数提高436.5倍.[结论]采用差速离心与脉冲电泳法结合可以有效地从感染WBD长春花中分离到纯的WBD植原体染色体DNA,WBD植原体染色体DNA大小约为650 kb.     

Shotgun proteomic profiling of five species of New Zealand Pachycladon

The genus Pachycladon consists of ten species of alpine plants, nine of which are endemic to New Zealand. The species are closely related to the model plant Arabidopsis thaliana with respect to their sequence divergence and chromosome synteny, occupy distinct geographical habitats in terms of both latitude and altitude, and display a range of morphologies. We have performed label‐free quantitative shotgun proteomic analysis of five different species of Pachycladon, namely P. cheesemanii (CH), P. exile (EX), P. fastigiatum (FA), P. enysii (EN) and P. novae‐zelandiae (NZ). The total non‐redundant data set for all five species contained 1489 proteins. The numbers of proteins identified reproducibly in each species ranged from 629 for CH to 987 for NZ, with 681 for EN, 741 for EX and 934 for FA. Previous metabolite‐based studies have shown that FA hydrolyzes glucosinolates completely to isothiocyanates while EN converts glucosinolates to nitriles. In this study, we observed high expression of ESP (At1g54040, epithiospecifying senescence regulator protein) and myrosinase 2 (At5g25980, glycosyl hydrolase family protein), which result in production of nitriles and epithionitriles, in EN and NZ, and we also observed higher expression of ESM1 (At3g14210, GDSL esterase/lipase), which mediates the formation of isothiocyanate, in FA.     

Shotgun proteomic analysis of wing discs from the domesticated silkworm (Bombyx mori) during metamorphosis

Proteomic profiles from the wing discs of silkworms at the larval, pupal, and adult moth stages were determined using shotgun proteomics and MS sequencing. We identified 241, 218, and 223 proteins from the larval, pupal, and adult moth stages, respectively, of which 139 were shared by all three stages. In addition, there were 55, 37, and 43 specific proteins identified at the larval, pupal, and adult moth stages, respectively. More metabolic enzymes were identified among the specific proteins expressed in the wing disc of larvae compared with pupae and moths. The identification of FKBP45 and the chitinase-like protein EN03 as two proteins solely expressed at the larval stage indicate these two proteins may be involved in the immunological functions of larvae. The myosin heavy chain was identified in the pupal wing disc, suggesting its involvement in the formation of wing muscle. Some proteins, such as proteasome alpha 3 subunits and ribosomal proteins, specifically identified from the moth stage may be involved in the degradation of old cuticle proteins and new cuticle protein synthesis. Gene ontology analysis of proteins specific to each of these three stages enabled their association with cellular component, molecular function, and biological process categories. The analysis of similarities and differences in these identified proteins will greatly further our understanding of wing disc development in silkworm and other insects.     

Metabolomic analysis reveals the potential metabolites and pathogenesis involved in mulberry yellow dwarf disease

To analyse the molecular mechanisms of phytoplasma pathogenicity, the comprehensive metabolomic changes of mulberry leaf and phloem sap in response to phytoplasma infection were examined using gas chromatography‐mass spectrometry. The metabolic profiles obtained revealed that the metabolite compositions of leaf and phloem sap were different, and phytoplasma infection has a greater impact on the metabolome of phloem sap than of leaf. Phytoplasma infection brought about the content changes in various metabolites, such as carbohydrates, amino acids, organic acids, etc. Meanwhile, the results of biochemical analysis showed that the degradation of starch was repressed, and the starch content was increased in the infected leaves. In addition, we found that phytoplasma infection changed the levels of abscisic acid and cytokinin and break phytohormone balance. Interestingly, our data showed that the contents of H₂O₂ and superoxide were increased in the infected leaves, but not in the phloem saps. Based on the results, the expression levels of the genes involved in the metabolism of some changed metabolites were examined, and the potential molecular mechanisms of these changes were discussed. It can be concluded that both the leaf and phloem saps have a complicated metabolic response to phytoplasma infection, but their response mechanisms were different.     

Shotgun proteomic analysis of Bombyx mori brain: emphasis on regulation of behavior and development of the nervous system

The insect brain plays crucial roles in the regulation of growth and development and in all types of behavior. We used sodium dodecyl sulfate polyacrylamide gel electrophoresis and high‐performance liquid chromatography ? electron spray ionization tandem mass spectrometry (ESI‐MS/MS) shotgun to identify the proteome of the silkworm brain, to investigate its protein composition and to understand their biological functions. A total of 2210 proteins with molecular weights in the range of 5.64–1539.82 kDa and isoelectric points in the range of 3.78–12.55 were identified. These proteins were annotated according to Gene Ontology Annotation into the categories of molecular function, biological process and cellular component. We characterized two categories of proteins: one includes behavior‐related proteins involved in the regulation of behaviors, such as locomotion, reproduction and learning; the other consists of proteins related to the development or function of the nervous system. The identified proteins were classified into 283 different pathways according to KEGG analysis, including the PI3K‐Akt signaling pathway which plays a crucial role in mediating survival signals in a wide range of neuronal cell types. This extensive protein profile provides a basis for further understanding of the physiological functions in the silkworm brain.     

Shotgun proteomic analysis of the Bombyx mori anterior silk gland: An insight into the biosynthetic fiber spinning process

The Bombyx mori anterior silk gland (ASG) is a natural fiber manipulator for the material provided by the middle and posterior silk glands. In view of the significant role of the ASG in the liquid–crystal spinning process, a shotgun proteomics approach was taken to study the relationship between the function of proteins in the silkworm ASG and the spinning mechanism. A total of 1132 proteins with 7647 unique peptides were identified in the ASG dataset including some involved in the cuticle, ion transportation, energy metabolism, and apoptosis. Two putative cuticle‐specific proteins were highly and specifically expressed in the ASG; therefore, the ASG dataset could provide clues for comprehensive understanding of the natural silk spinning mechanism in the silkworm. All MS data have been deposited in the ProteomeXchange with identifier PXD000090.     

Cardiovascular proteomic analysis

Here, we report on our proteomic studies in the field of cardiovascular medicine. Our research has been focused on understanding the role of proteins in cardiovascular disease with a particular focus on epigenetic regulation and biomarker discovery, with the objective of better understanding cardiovascular pathophysiology to lead to the development of new and better diagnostic and therapeutic methods. We have used mass spectrometry for over 5 years as a viable method to investigate protein-protein interactions and post-translational modifications in cellular proteins as well as a method to investigate the role of extra-cellular proteins. Use of mass spectrometry not only as a research tool but also as a potential diagnostic tool is a topic of interest. In addition to these functional proteomics studies, structural proteomic studies are also done with expectations to allow for pinpoint drug design and therapeutic intervention. Collectively, our proteomics studies are focused on understanding the functional role and potential therapeutically exploitable property of proteins in cardiovascular disease from both intra-cellular and extra-cellular aspects with both functional as well as structural proteomics approaches to allow for comprehensive analysis.     

Shotgun lipidomics: multidimensional MS analysis of cellular lipidomes

Shotgun lipidomics, comprised of intrasource separation, multidimensional mass spectrometry and computer-assisted array analysis, is an emerging powerful technique in lipidomics. Through effective intrasource separation of predetermined groups of lipid classes based on their intrinsic electrical propensities, analyses of lipids from crude extracts of biologic samples can be directly and routinely performed. Appropriate multidimensional array analysis of lipid pseudomolecular ions and fragments can be performed leading to the identification and quantitation of targeted lipid molecular species. Since most biologic lipids are linear combinations of aliphatic chains, backbones and head groups, a rich repertoire of multiple lipid building blocks present in discrete combinations represent experimental observables that can be computer reconstructed in conjunction with their pseudomolecular ions to directly determine the lipid molecular structures from a lipid extract. Through this approach, dramatic increases in the accessible dynamic range for ratiometric quantitation and discrimination of isobaric molecular species can be achieved without any prior column chromatography or operator-dependent supervision. At its current state of development, shotgun lipidomics can analyze over 20 lipid classes, hundreds of lipid molecular species and more than 95% of the mass content of a cellular lipidome. Thus, understanding the biochemical mechanisms underlying lipid-mediated disease states will be greatly facilitated by the power of shotgun lipidomics.     

Shotgun proteome analysis of protein cleavage in apoptotic cells

A new shotgun proteomics approach was employed to identify degraded proteins. Jurkat T-cells were induced to undergo apoptosis by Fas (CD95/Apo-1) stimulation. The proteins were separated by large (30 cm) sodium dodecyl sulphate-polyacrylamide gel electrophoresis and identified by liquid chromatography-tandem mass spectrometry after digestion of 100 gel slices with trypsin. The molecular masses of the individual gel slices were calculated through the known theoretical masses of the identified proteins. Proteins were defined as degradation candidates if either the empirical determined molecular mass was at most 80% of the theoretical value, or if proteins were identified in clearly different gel slices. In this manner, the degradation of 11 already identified apoptosis-modified proteins was confirmed and nine until now unknown degradation candidate proteins identified. Degradation during apoptosis must be verified by additional techniques such as in vitro caspase assays as shown for nucleolin and Rho GDI 2. The results presented confirm the suitability of a shotgun approach for the identification of putative protease targets.     

Comparative proteomic analysis between the domesticated silkworm (Bombyx mori) reared on fresh mulberry leaves and on artificial diet

To gain an insight into the effects of different diets on growth and development of the domesticated silkworm at protein level, we employed comparative proteomic approach to investigate the proteomic differences of midgut, hemolymph, fat body and posterior silk gland of the silkworms reared on fresh mulberry leaves and on artificial diet. Seventy-six differentially expressed proteins were identified by MALDI TOF/TOF MS, and among them, 41 proteins were up-regulated, and 35 proteins were downregulated. Database searches, combined with GO analysis and KEGG pathway analysis revealed that some hemolymph proteins such as Nuecin, Gloverin-like proteins, PGRP, P50 and beta/-N-acetylglucosamidase were related to innate immunity of the silkworm, and some proteins identified in silkworm midgut including Myosin 1 light chain, Tropomyosin 1, Profilin, Serpin-2 and GSH-Px were involved in digestion and nutrition absorption. Moreover, two up-regulated enzymes in fat body of larvae reared on artificial diet were identified as V-ATPase subunit B and Arginine kinase which participate in energy metabolism. Furthermore, 6 down-regulated proteins identified in posterior silk gland of silkworm larvae reared on artificial diet including Ribosomal protein SA, EF-2, EF-1gamma, AspAT, ERp57 and PHB were related to silk synthesis. Our results suggested that the different diets could alter the expression of proteins related to immune system, digestion and absorption of nutrient, energy metabolism and silk synthesis poor nutrition and absorption of nutrition in silkworm. The results also confirmed that the poor nutrient absorption, weakened innate immunity, decreased energy metabolism and reduced silk synthesis are the main reasons for low cocoons yield, inferior filament quality, low survival rate of young larvae and insufficient resistance against specific pathogens in the silkworms fed on artificial diet.