共查询到20条相似文献,搜索用时 15 毫秒
1.
To establish a proteomic reference map for soybean leaves, we separated and identified leaf proteins using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and mass spectrometry (MS). Tryptic digests of 260 spots were subjected to peptide mass fingerprinting (PMF) by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) MS. Fifty-three of these protein spots were identified by searching NCBInr and SwissProt databases using the Mascot search engine. Sixty-seven spots that were not identified by MALDI-TOF-MS analysis were analyzed with liquid chromatography tandem mass spectrometry (LC-MS/MS), and 66 of these spots were identified by searching against the NCBInr, SwissProt and expressed sequence tag (EST) databases. We have identified a total of 71 unique proteins. The majority of the identified leaf proteins are involved in energy metabolism. The results indicate that 2D-PAGE, combined with MALDI-TOF-MS and LC-MS/MS, is a sensitive and powerful technique for separation and identification of soybean leaf proteins. A summary of the identified proteins and their putative functions is discussed. 相似文献
2.
A proteomic analysis of bovine amniotic and allantoic fluids collected around Day 45 of gestation was performed using gel-based and LC-based MS workflows. A depletion/enrichment protocol using ultrafiltration under denaturing and reducing conditions produced an enriched fraction containing protein species predominantly between 5 and 50 kDa molecular weight. The analyses of conceptus fluid proteins were performed using two strategies; first, 2-DE coupled with MALDI-TOF-MS/MS and LC-ESI-MS/MS analysis of individual protein spots and second, a global protein snapshot of the enriched 5-50 kDa protein fraction by LC-ESI-MS/MS and LC-MALDI-TOF-MS/MS. Allocation of bovine specific protein identities was achieved by searching the Interactive Bovine In Silico SNP (IBISS) and NCBInr protein sequence databases resulting in the confident PMF identification and MS/MS confirmation of >200 2-DE generated allantoic fluids protein spots (74 individual protein species identified) and the MS/MS peptide identification of 105 LC-ESI-MS/MS generated protein identities. In total, the identity of 139 individual protein species from allantoic fluids was confirmed with peptide sequence probability MOWSE scores at the p<0.05 level or better. The comparison of bovine Day 45 amniotic and allantoic fluids protein profiles revealed differences between these two conceptus fluids in early pregnancy. 相似文献
3.
Panthangi Seshapani Daddam Jayasimha Rayalu Vadde Kiran Kumar Kathera Chandra Sekhar Jasti Pramoda Kumari 《Bioinformation》2013,9(9):485-490
Differently expressed proteins in probiotic Escherichia coli nissle 1917 under mercury stress identified by using a proteomic
approach. We applied to separate proteins by using two-dimensional gel electrophoresis and proteins were identified using
MALDI-TOF-MS using PMF, by mascot database search using the NCBI database. we identified six proteins after exposure to
mercury stress with respect to different functional classes. It is useful to understand the molecular insights into mercury stress in
probiotic E. coli. Next we describe a structure generated by homology modelling and functional domain identification; it is
interesting to study the impact of stress on protein structures. MS characterization and computational methods together provide
the opportunity to examine the impact of stress arising from mercury. The role of these proteins in metal tolerance and structure
relation is discussed. To the best of our knowledge, proteomics of E. coli nissle 1917 overview of mercury stress has been reported
for the first time. 相似文献
4.
Zhou H Wang F Wang Y Ning Z Hou W Wright TG Sundaram M Zhong S Yao Z Figeys D 《Molecular & cellular proteomics : MCP》2011,10(10):O111.008425
Despite their importance in many biological processes, membrane proteins are underrepresented in proteomic analysis because of their poor solubility (hydrophobicity) and often low abundance. We describe a novel approach for the identification of plasma membrane proteins and intracellular microsomal proteins that combines membrane fractionation, a centrifugal proteomic reactor for streamlined protein extraction, protein digestion and fractionation by centrifugation, and high performance liquid chromatography-electrospray ionization-tandem MS. The performance of this approach was illustrated for the study of the proteome of ER and Golgi microsomal membranes in rat hepatic cells. The centrifugal proteomic reactor identified 945 plasma membrane proteins and 955 microsomal membrane proteins, of which 63 and 47% were predicted as bona fide membrane proteins, respectively. Among these proteins, >800 proteins were undetectable by the conventional in-gel digestion approach. The majority of the membrane proteins only identified by the centrifugal proteomic reactor were proteins with ≥ 2 transmembrane segments or proteins with high molecular mass (e.g. >150 kDa) and hydrophobicity. The improved proteomic reactor allowed the detection of a group of endocytic and/or signaling receptor proteins on the plasma membrane, as well as apolipoproteins and glycerolipid synthesis enzymes that play a role in the assembly and secretion of apolipoprotein B100-containing very low density lipoproteins. Thus, the centrifugal proteomic reactor offers a new analytical tool for structure and function studies of membrane proteins involved in lipid and lipoprotein metabolism. 相似文献
5.
MS/MS techniques in proteomics make possible the identification of proteins from organisms with little or no genome sequence information available. Peptide sequences are obtained from tandem mass spectra by matching peptide mass and fragmentation information to protein sequence information from related organisms, including unannotated genome sequence data. This peptide identification data can then be grouped and reconstructed into protein data. In this study, we have used this approach to study protein secretion by Aspergillus flavus, a filamentous fungus for which very little genome sequence information is available. A. flavus is capable of degrading the flavonoid rutin (quercetin 3-O-glycoside), as the only source of carbon via an extracellular enzyme system. In this continuing study, a proteomic analysis was used to identify secreted proteins from A. flavus when grown on rutin. The growth media glucose and potato dextrose were used to identify differentially expressed secreted proteins. The secreted proteins were analyzed by 1- and 2-DE and MS/MS. A total of 51 unique A. flavus secreted proteins were identified from the three growth conditions. Ten proteins were unique to rutin-, five to glucose- and one to potato dextrose-grown A. flavus. Sixteen secreted proteins were common to all three media. Fourteen identifications were of hypothetical proteins or proteins of unknown functions. To our knowledge, this is the first extensive proteomic study conducted to identify the secreted proteins from a filamentous fungus. 相似文献
6.
Glen A Gan CS Hamdy FC Eaton CL Cross SS Catto JW Wright PC Rehman I 《Journal of proteome research》2008,7(3):897-907
The unpredictable behavior of prostate cancer presents a major clinical challenge during patient management. In order to gain an insight into the molecular mechanisms associated with prostate cancer progression, we employed the shot-gun proteomic approach of isobaric tags for relative and absolute quantitation (iTRAQ), followed by 2D-LC-MS/MS, using the poorly metastatic LNCaP cell line and its highly metastatic variant LNCaP-LN3 cell line as a model. A total number of 280 unique proteins were identified (> or =95% confidence), and relative expression data was obtained for 176 of these. Ten proteins were found to be significantly up-regulated (> or =1.50 fold), while 4 proteins were significantly down-regulated (> or = -1.50 fold), in LNCaP-LN3 cells. Differential expression of brain creatine kinase (CKBB), soluble catechol-O-methyltransferase (S-COMT), tumor rejection antigen (gp96), and glucose regulated protein, 78 kDa (grp78), was confirmed by Western blotting or independent 2D-PAGE analysis. Additionally, iTRAQ analysis identified absence of the lactate dehydrogenase-B (LDH-B) subunit in LNCaP-LN3 cells, confirming our published data. The clinical relevance of gp96 was assessed by immunohistochemistry using prostate tissues from benign ( n = 95), malignant ( n = 66), and metastatic cases ( n = 3). Benign epithelium showed absent/weak gp96 expression in the basal cells, in contrast to the moderate/strong expression seen in malignant epithelium. Furthermore, there was a statistically significant difference in the intensity of gp96 expression between benign and malignant cases ( p < 0.0005, Mann-Whitney U). Our study is the first to report the application of iTRAQ technology and its potential for the global proteomic profiling of prostate cancer cells, including the identification of absent protein expression. 相似文献
7.
Ostrowski LE Blackburn K Radde KM Moyer MB Schlatzer DM Moseley A Boucher RC 《Molecular & cellular proteomics : MCP》2002,1(6):451-465
Cilia play an essential role in protecting the respiratory tract by providing the force necessary for mucociliary clearance. Although the major structural components of human cilia have been described, a complete understanding of cilia function and regulation will require identification and characterization of all ciliary components. Estimates from studies of Chlamydomonas flagella predict that an axoneme contains > or = 250 proteins. To identify all the components of human cilia, we have begun a comprehensive proteomic analysis of isolated ciliary axonemes. Analysis by two-dimensional (2-D) PAGE resulted in a highly reproducible 2-D map consisting of over 240 well resolved components. Individual protein spots were digested with trypsin and sequenced using liquid chromatography/tandem mass spectrometry (LC/MS/MS). Peptide matches were obtained to 38 potential ciliary proteins by this approach. To identify ciliary components not resolved by 2-D PAGE, axonemal proteins were separated on a one-dimensional gel. The gel lane was divided into 45 individual slices, each of which was analyzed by LC/MS/MS. This experiment resulted in peptide matches to an additional 110 proteins. In a third approach, preparations of isolated axonemes were digested with Lys-C, and the resulting peptides were analyzed directly by LC/MS/MS or by multidimensional LC/MS/MS, leading to the identification of a further 66 proteins. Each of the four approaches resulted in the identification of a subset of the proteins present. In total, sequence data were obtained on over 1400 peptides, and over 200 potential axonemal proteins were identified. Peptide matches were also obtained to over 200 human expressed sequence tags. As an approach to validate the mass spectrometry results, additional studies examined the expression of several identified proteins (annexin I, sperm protein Sp17, retinitis pigmentosa protein RP1) in cilia or ciliated cells. These studies represent the first proteomic analysis of the human ciliary axoneme and have identified many potentially novel components of this complex organelle. 相似文献
8.
Kristin LM Boylan Somaieh Afiuni-Zadeh Melissa A Geller Kayla Hickey Timothy J Griffin Stefan E Pambuccian Amy PN Skubitz 《Clinical proteomics》2014,11(1):30
Background
The proteomic analysis of body fluids is a growing technology for the identification of protein biomarkers of disease. Given that Papanicolaou tests (Pap tests) are routinely performed on over 30 million women annually in the U.S. to screen for cervical cancer, we examined the residual Pap test fluid as a source of protein for analysis by mass spectrometry (MS). In the liquid-based Pap test, cervical cells are collected from the ectocervix and placed into an alcohol-based fixative prior to staining and pathologic examination. We hypothesized that proteins shed by cells of the female genital tract can be detected in the Pap test fixative by MS-based proteomic techniques. We examined the feasibility of using residual fluid from discarded Pap tests with cytologically “normal” results to optimize sample preparation for MS analysis. The protein composition of the cell-free Pap test fluid was determined by silver staining of sodium dodecyl sulfate -polyacrylamide gels, and the abundance of serum proteins was examined by Western immunoblot using an antibody against human serum albumin. Both pooled and individual samples were trypsin digested and analyzed by two-dimensional MS/MS. Proteins were identified by searching against the Human Uniprot database, and characterized for localization, function and relative abundance.Results
The average volume of the residual Pap test fluid was 1.5 ml and the average protein concentration was 0.14 mg/ml. By Western immunoblot we showed that the amount of albumin in each sample was significantly reduced compared to normal serum. By MS/MS, we identified 714 unique proteins in pooled Pap test samples and an average of 431 proteins in individual samples. About 40% of the proteins identified were extracellular or localized to the plasma membrane. Almost 20% of the proteins identified were involved in immunity and defense, characteristic of the healthy cervical-vaginal proteome. By merging the protein sets from the individual and pooled Pap test samples, we created a “Normal Pap test Core Proteome” consisting of 153 proteins.Conclusions
Residual Pap test fluid contains a sufficient amount of protein for analysis by MS and represents a valuable biospecimen source for the identification of protein biomarkers for gynecological diseases. 相似文献9.
A proteomic approach has been adopted to investigate the low-abundant proteins in rice leaf in response to cold stress. Rice seedlings were exposed to different temperatures, such as 5 or 10 degrees C, and samples were collected after different time course. To eliminate the high-abundant proteins in leaf tissues such as ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), proteins were fractionated by polyethylene glycol (PEG). The elimination of Rubisco from the protein samples was confirmed by Western blot analysis. The PEG fractionated protein samples were separated by 2-DE and visualized by silver or CBB staining. A total 12 up-regulated protein spots were identified using the analysis of MALDI-TOF mass spectrometry or ESI MS/MS. We identified some novel proteins such as cysteine proteinase, thioredoxin peroxidase, a RING zinc finger protein-like, drought-inducible late embryogenesis abundant, and a fibrillin-like protein that had not yet been reported in the earlier reports on cold proteomic analysis. The identification of some novel low-abundant proteins in response to cold stress may provide a new homeostasis to develop enhanced cold tolerance transgenic plants. Thus, we propose that a PEG fractionation system can be used as an influential protein extraction method from the leaf samples, which can lead to knowledge of the expression pattern of low-abundant proteins in response to various biotic or abiotic stresses. 相似文献
10.
Serum analysis represents an extreme challenge due to the dynamic range of the proteins of interest, and the high structural complexity of the constituent proteins. In serum, the quantities of proteins and peptides of interest range from those considered "high abundance", present at 2-70% by mass of total protein, to those considered "low abundance", present at 10(-12) M or less. This range of analytical target molecules is outside the realm of available technologies for proteomic analysis. Therefore, in this study, we have developed a workflow toward addressing the complexity of these samples through the application of multidimensional separation techniques. The use of reversed-phase methods for the separation and fractionation of protein samples has been investigated, with the goal of developing an optimized serum separation for application to proteomic analysis. Samples of human serum were depleted of the six most abundant proteins, using an immunoaffinity LC method, then were separated under a variety of reversed-phase (RP) conditions using a macroporous silica C18 surface modified column material. To compare the qualities of the RP separations of this complex protein sample, absorbance chromatograms were compared, and fractions were collected for off-line SDS-PAGE and 2D-LC-MS/MS analysis. The column fractions were further investigated by determination of protein identities using either whole selected fractions, or gel bands excised from SDS-PAGE gels of the fractions. In either case samples underwent tryptic fragmentation and peptide analysis using MALDI-MS or LC-MS/MS. The preferred conditions for RP protein separation exhibited reproducibly high resolution and high protein recoveries (>98%, as determined by protein assay). Using the preferred conditions also permitted high column mass load, with up to 500 microg of protein well tolerated using a 4.6 mm ID x 50 mm column, or up to 1.5 mg on a 9.4 mm ID x 50 mm column. Elevated column temperature (80 degrees C) was observed to be a critical operational parameter, with poorer results observed at lower temperatures. The combination of sample simplification by immunoaffinity depletion combined with a robust and high recovery RP-HPLC fractionation yields samples permitting higher quality protein identifications by coupled LC-MS methods. 相似文献
11.
The goal of commercial yellow perch aquaculture is to increase muscle mass which leads to increased profitability. The accumulation and degradation of muscle-specific gene products underlies the variability in body mass (BM) and length observed in pond-cultured yellow perch. Our objective was to apply a combination of statistical and proteomic technologies to identify intact and/or proteolytic fragments of muscle specific gene products involved in muscle growth in yellow perch. Seventy yellow perch randomly selected at 10, 12, 16, 20, and 26 wk of age were euthanized; BM and length were measured and a muscle sample taken. Muscle proteins were resolved using 5-20% gradient SDS-PAGE, stained with SYPRO Ruby and analyzed using TotalLab software. Data were analyzed using stepwise multiple regression with the dependent variables, BM and length and proportional OD of each band in a sample as a potential regressor. Eight bands associated with BM (R(2) = 0.84) and nine bands with length (R(2) = 0.85) were detected. Protein sequencing by nano-LC/MS/MS identified 20 proteins/peptides associated with BM and length. These results contribute the identification of gene products and/or proteolytic fragments associated with muscle growth in yellow perch. 相似文献
12.
Because of their complexity, the separation of intact proteins from complex mixtures is an important step to comparative proteomics and the identification and characterization of the proteins by mass spectrometry (MS). In the study reported, we evaluated the use of nonporous-reversed-phase (np-RP)-HPLC for intact protein separation prior to MS analyses. The separation system was characterized and compared to 1D-SDS-PAGE electrophoresis in terms of resolution and sensitivity. We demonstrate that np-RP-HPLC protein separation is highly reproducible and provides intact protein fractions which can be directly analyzed by MALDI-TOF-MS for intact molecular weight determination. An in-well digestion protocol was developed, allowing for rapid protein identification by peptide mass fingerprinting (PMF) and resulted in comparable or improved peptide recovery compared with in-gel digestion. The np-RP sensitivity of detection by UV absorbance at 214 nm for intact proteins was at the low ng level and the sensitivity of peptide analysis by MALDI-TOF-MS was in the 10-50 fmol level. A membrane protein fraction was characterized to demonstrate application of this methodology. Among the identified proteins, multiple forms of vimentin were observed. Overall, we demonstrate that np-RP-HPLC followed by MALDI-TOF-MS allows for rapid, sensitive, and reproducible protein fractionation and very specific protein characterization by integration of PMF analysis with MS intact molecular weight information. 相似文献
13.
Rosiglitazone, one of the thiazolidinedione (TZD), is an oral antidiabetic drug that activates a gamma isoform of peroxisome
proliferator-activated receptor (PPARγ). To identify target proteins induced by rosiglitazone in adipocytes, we first performed
simultaneous in-depth proteomic profiling of cytosolic proteins and secreted proteins (secretome) from 3T3-L1 adipocytes using
a label-free quantification method with nano-UPLC MS/MS. In total, we identified 646 proteins from 3T3-L1 adipocytes, of which
172 and 162 proteins were upregulated and downregulated >1.5-fold, respectively, in rosiglitazone-treated cells, as compared
to controls. Some differentially expressed proteins in particular, including fatty acid translocase (FAT)/CD36, fatty acid
binding protein, lipoprotein lipase, acetyl CoA acyltransferase, carnitine O-palmitoyltransferase 2, sterol carrier protein,
adiponectin, and phosphoenolpyruvate carboxykinase could explain the current action mechanism of TZDs. Furthermore, this study
is the first to report on two potential target proteins of rosiglitazone, such as adenomatosis polyposis coli 2 (APC2), and
eukaryotic translation initiation factor 5A-1 (eIF5A) related to apoptosis and cell division. Our data clearly suggest that
in-depth proteomic approaches using cytosolic and secreted proteins are important and necessary for identification of drug
targets at the protein level. 相似文献
14.
Millares P Lacourse EJ Perally S Ward DA Prescott MC Hodgkinson JE Brophy PM Rees HH 《PloS one》2012,7(3):e33590
Lack of genomic sequence data and the relatively high cost of tandem mass spectrometry have hampered proteomic investigations into helminths, such as resolving the mechanism underpinning globally reported anthelmintic resistance. Whilst detailed mechanisms of resistance remain unknown for the majority of drug-parasite interactions, gene mutations and changes in gene and protein expression are proposed key aspects of resistance. Comparative proteomic analysis of drug-resistant and -susceptible nematodes may reveal protein profiles reflecting drug-related phenotypes. Using the gastro-intestinal nematode, Haemonchus contortus as case study, we report the application of freely available expressed sequence tag (EST) datasets to support proteomic studies in unsequenced nematodes. EST datasets were translated to theoretical protein sequences to generate a searchable database. In conjunction with matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF-MS), Peptide Mass Fingerprint (PMF) searching of databases enabled a cost-effective protein identification strategy. The effectiveness of this approach was verified in comparison with MS/MS de novo sequencing with searching of the same EST protein database and subsequent searches of the NCBInr protein database using the Basic Local Alignment Search Tool (BLAST) to provide protein annotation. Of 100 proteins from 2-DE gel spots, 62 were identified by MALDI-TOF-MS and PMF searching of the EST database. Twenty randomly selected spots were analysed by electrospray MS/MS and MASCOT Ion Searches of the same database. The resulting sequences were subjected to BLAST searches of the NCBI protein database to provide annotation of the proteins and confirm concordance in protein identity from both approaches. Further confirmation of protein identifications from the MS/MS data were obtained by de novo sequencing of peptides, followed by FASTS algorithm searches of the EST putative protein database. This study demonstrates the cost-effective use of available EST databases and inexpensive, accessible MALDI-TOF MS in conjunction with PMF for reliable protein identification in unsequenced organisms. 相似文献
15.
A novel approach and protocol for discovering extremely low-abundance proteins in serum 总被引:1,自引:0,他引:1
Tanaka Y Akiyama H Kuroda T Jung G Tanahashi K Sugaya H Utsumi J Kawasaki H Hirano H 《Proteomics》2006,6(17):4845-4855
The proteomic analysis of serum (plasma) has been a major approach to determining biomarkers essential for early disease diagnoses and drug discoveries. The determination of these biomarkers, however, is analytically challenging since the dynamic concentration range of serum proteins/peptides is extremely wide (more than 10 orders of magnitude). Thus, the reduction in sample complexity prior to proteomic analyses is essential, particularly in analyzing low-abundance protein biomarkers. Here, we demonstrate a novel approach to the proteomic analyses of human serum that uses an originally developed serum protein separation device and a sequentially linked 3-D-LC-MS/MS system. Our hollow-fiber-membrane-based serum pretreatment device can efficiently deplete high-molecular weight proteins and concentrate low-molecular weight proteins/peptides automatically within 1 h. Four independent analyses of healthy human sera pretreated using this unique device, followed by the 3-D-LC-MS/MS successfully produced 12 000-13 000 MS/MS spectra and hit around 1800 proteins (>95% reliability) and 2300 proteins (>80% reliability). We believe that the unique serum pretreatment device and proteomic analysis protocol reported here could be a powerful tool for searching physiological biomarkers by its high throughput (3.7 days per one sample analysis) and high performance of finding low abundant proteins from serum or plasma samples. 相似文献
16.
A proteomic approach to identification of transmembrane proteins and membrane-anchored proteins of Arabidopsis thaliana by peptide sequencing. 总被引:2,自引:0,他引:2
Takashi Sazuka Sumie Keta Katsuhiro Shiratake Shohei Yamaki Daisuke Shibata 《DNA research》2004,11(2):101-113
A proteomic approach was developed for the identification of membrane-bound proteins of Arabidopsis thaliana. A subcellular fraction enriched in vacuolar membranes was prepared from 4-week-old plants and was washed with various agents to remove peripheral membrane proteins and contaminating soluble proteins. The remaining membrane-bound proteins were then subjected to proteomic analysis. Given that these proteins were resolved poorly by standard two-dimensional gel electrophoresis, we subjected them instead to SDS-polyacrylamide gel electrophoresis and to protein digestion within gel slices with lysylendopeptidase. The resulting peptides were separated by reverse-phase high-performance liquid chromatography and subjected to Edman sequencing. From the 163 peptide peaks analyzed, 69 peptide sequences were obtained, 64 of which were informative. The proteins corresponding to these peptide sequences were identified as belonging to 42 families, including two subfamilies, by comparison with the protein sequences predicted from annotation of the A. thaliana genome. A total of 34 proteins was identified definitively with protein-specific peptide sequences. Transmembrane proteins detected in the membrane fraction included transporters, channels, receptors, and unknown molecules, whereas the remaining proteins, categorized as membrane-anchored proteins, included small GTPases, GTPase binding proteins, heat shock protein 70-like proteins, ribosomal proteins, and unknown proteins. These membrane-anchored proteins are likely attached to membranes by hydrophobic anchor molecules or through tight association with other membrane-bound proteins. This proteomic approach has thus proved effective for the identification of membrane-bound proteins. 相似文献
17.
Proteomic analysis of maternal serum in down syndrome: identification of novel protein biomarkers 总被引:2,自引:0,他引:2
Nagalla SR Canick JA Jacob T Schneider KA Reddy AP Thomas A Dasari S Lu X Lapidus JA Lambert-Messerlian GM Gravett MG Roberts CT Luthy D Malone FD D'Alton ME 《Journal of proteome research》2007,6(4):1245-1257
Down syndrome (DS) is the most prevalent chromosomal disorder, accounting for significant morbidity and mortality. Definitive diagnosis requires invasive amniocentesis, and current maternal serum-based testing requires a false-positive rate of about 5% to detect 85% of affected pregnancies. We have performed a comprehensive proteomic analysis to identify potential serum biomarkers to detect DS. First- and second-trimester maternal serum samples of DS and gestational age-matched controls were analyzed using multiple, complementary proteomic approaches, including fluorescence 2-dimensional gel electrophoresis (2D-DIGE), 2-dimensional liquid chromatography-chromatofocusing (2D-CF), multidimensional protein identification technology (MudPIT; LC/LC-MS/MS), and MALDI-TOF-MS peptide profiling. In total, 28 and 26 proteins were differentially present in first- and second-trimester samples, respectively. Of these, 19 were specific for the first trimester and 16 for the second trimester, and 10 were differentially present in both trimesters. Analysis of MALDI-TOF-MS peptide profiles with pattern-recognition software also discriminated between DS and controls in both trimesters, with an average recognition capability approaching 96%. A majority of the biomarkers identified are serum glycoproteins that may play a role in cellular differentiation and growth of fetus. Further characterization and quantification of these markers in a larger cohort of subjects may provide the basis for new tests for improved DS screening. 相似文献
18.
Proteomic analysis of the excretory-secretory proteins of the Trichinella spiralis L1 larva, a nematode parasite of skeletal muscle 总被引:2,自引:0,他引:2
Trichinella spiralis is an intracellular nematode parasite of mammalian skeletal muscle. Infection of the muscle cell leads to the formation of a host-parasite complex that results in profound alterations to the host cell and a re-alignment of muscle-specific gene expression. The role of parasite excretory-secretory (ES) proteins in mediating these effects is currently unknown, largely due to the difficulty in identifying and assigning function to individual proteins. In this study, a global proteomics approach was used to analyse the ES proteins from T. spiralis muscle larvae. Following 2-DE of ES proteins,MALDI-TOF-MS and LC-MS/MS were used to identify the peptide spots. Specific Trichinella EST databases were assembled and used to analyse the data. Despite the current absence of a Trichinella genome-sequencing project, 43 out of 52 protein spots analysed were identified and included the major secreted glycoproteins. Other novel proteins were identified from matches with sequences in the T. spiralis database. Our results demonstrate the value of proteomics as a tool for the identification of Trichinella ES proteins and in the study of the molecular mechanism underpinning the formation of the host-parasite complex during Trichinella infections. 相似文献
19.
Rodrigues SP Ventura JA Aguilar C Nakayasu ES Almeida IC Fernandes PM Zingali RB 《Proteomics》2011,11(13):2592-2602
Papaya (Carica papaya L.) hosts the only described laticifer-infecting virus (Papaya meleira virus, PMeV), which is the causal agent of papaya sticky disease. To understand the systemic effects of PMeV in papaya, we conducted a comprehensive proteomic analysis of leaf samples from healthy and diseased plants grown under field conditions. First, a reference 2-DE map was established for proteins from healthy samples. A total of 486 reproducible spots were identified, and MALDI-TOF-MS/MS data identified 275 proteins accounting for 159 distinct proteins from 231 spots that were annotated. Second, the differential expression of proteins from healthy and diseased leaves was determined through parallel experiments, using 2-DE and DIGE followed by MALDI-TOF-MS/MS and LC-IonTrap-MS/MS, respectively. Conventional 2-DE analysis revealed 75 differentially expressed proteins. Of those, 48 proteins were identified, with 26 being upregulated (U) and 22 downregulated (D). In general, metabolism-related proteins were downregulated, and stress-responsive proteins were upregulated. This expression pattern was corroborated by the results of the DIGE analysis, which identified 79 differentially expressed proteins, with 23 identified (17 U and 6 D). Calreticulin and the proteasome subunits 20S and RPT5a were shown to be upregulated during infection by both 2-DE and DIGE analyses. These data may help shed light on plant responses against stresses and viral infections. 相似文献
20.
Iftekhar Alam Shamima Akhtar Sharmin Kyung-Hee Kim Yong-Goo Kim Jeung Joo Lee Jeong Dong Bahk Byung-Hyun Lee 《Plant and Soil》2011,346(1-2):45-62
Salinity together with waterlogging or flooding, a condition that occurs frequently in the field, can cause severe damage to crops. Combined flooding and salinity decreases the growth and survival of plants more than either stress alone. We report here the first proteomic analysis to investigate the global effects of saline flooding on multiple metabolic pathways. Soybean seedlings at the emergence (VE) stage were treated with 100 mM NaCl and flooded with water or 100 mM sodium chloride solution for 2 days. Proteins were extracted from hypocotyl and root samples and analyzed by two-dimensional gel electrophoresis followed by MALDI-TOF, MALDI-TOF/TOF mass spectrometry or immunoblotting. A total of 43 reproducibly resolved, differentially expressed protein spots visualized by Coomassie brilliant blue staining were identified by MALDI-TOF MS. Identities of several proteins were also validated by MS/MS analysis or immunoblot analysis. Twenty-nine proteins were upregulated, eight proteins were downregulated and six spots were newly induced. The identified proteins include well-known salt and flooding induced proteins as well as novel proteins expressed by the salinity-flooding combined stress. The comparative analysis identified changes at the proteome level that are both specific and part of a common or shared response. The identification of such differentially expressed proteins provides new targets for future studies that will allow assessment of their physiological roles and significance in the response of glycophytes to a combination of flooding and salinity. 相似文献