Tissue matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS)may identify lipids differentially expressed between cancer and adjacent normal tissue.To identify lipidomic profiles for gastric cancer, 24 gastric cancerswere profiled for lipid by the histology-directed, tissue MALDI MS technology.Lipid profiles differed between gastric cancer and adjacent normal tissue samples.At P<0.05, median class prediction accuracy in 100 random training-to-test partitions was 83.3% (5/6) for all classifiers tested.A peak at m/z 741.6 (sphingomyelin 34:1 K) was overexpressed, and a peak at m/z 782.6 (phosphatidylcholine 34:1 Na) was underexpressed in gastric cancers compared with normal tissue.Thus, lipid MALDI MS analysis may capture a global alteration in lipid profile of gastric cancer tissue, distinguishing cancerous epithelium from normal epithelium.
Chronic pancreatitis (CP) is currently diagnosed using invasive endoscopic and imaging techniques. However, urine can be collected safely and noninvasively and as such may offer a superior alternative to current techniques of CP diagnosis. We use mass spectrometry-based methods to discover proteins which are exclusive to or differentially abundant in urine of chronic pancreatitis patients. We have performed a comparative quantitative proteomic analysis of urine collected from 5 healthy controls, 5 severe CP patients, and 5 patients of a mixed cohort with clinical representation typical of patients referred for CP, but not diagnosed with the disease. Proteins from urine were fractionated via SDS-PAGE and digested in-gel with trypsin prior to reversed-phase liquid chromatography in-line with a mass spectrometer. ProteinPilot software and the QSPEC algorithm identified proteins and determined statistically significant differences between cohorts. We identified over 600 proteins from urine, of which several hundred were either exclusive to or differ quantitatively in severe CP patients. Members of the cathepsin protein family were of significantly higher abundance in the severe CP co-hort. In addition, we have identified a core set of 50 proteins in all 15 samples, 25 of which showed no significant difference among the cohorts. The differentially abundant proteins in severe CP patients represent an initial set of targets for directed proteomics experiments for further validation studies.However, larger matched cohorts will be required to determine if these differences have statistically significant diagnostic potential.
Background: Our previous microarray study showed that sevoflurane anesthesia affects the expression of rat genes in multiple organs including the liver. In this study, we investigated whether liver protein expression was altered after propofol, sevoflurane, or isoflurane anesthesia. We also investigated differences in the time course of each drug 24 and 72 h after anesthesia. Methods: Rats were randomly assigned to four groups (non-anesthetized group and three groups anesthetized at each time point, n = 6 per group). A venous catheter was inserted into the caudal vein of all rats. Rats were anesthetized with each agent for 6 h, and the liver was obtained immediately after anesthesia. Proteomic analysis was performed. Results: About 4200 spots in each gel were discriminated, and at least 2619 spots were matched. Using LC-MS/MS, we identified 47 spots for propofol, 45 spots for sevoflurane, and 21 spots for isoflurane that were differentially expressed (p < 0.05) 0 h after anesthesia. The numbers of altered proteins were 14 and 19 in the isoflurane and sevoflurane groups, respectively, 72 h after anesthesia, but alterations in 40 proteins were seen in the propofol group 72 h after anesthesia. Conclusion: Volatile and intravenous anesthetics affected protein expression in the liver. Alterations were different for each drug, with isoflurane showing fewer altered proteins 0 h after anesthesia than the other two drugs. The time courses of those proteins were also different between individual anesthetics, suggesting fewer alterations in rat liver protein expression with volatile anesthetics than with propofol.
A two-dimensional gel electrophoresis (2D-GE) and capillary isoelectric focusing (cIEF) were performed subsequently to analyze protein mixtures from laboratory animal tissues (liver, muscle, and mammary gland).Both techniques were compared and evaluated.It was found that cIEF can be a valuable alternative in comparison to 2D-GE.Especially considering the goals associated with the determination of far the separated proteins, cIEF possesses perspective analytical advantages.
Differences in serum protein profiles were analyzed to identify possible biomarkers associated with a poultry leg problem named tibial dyschondroplasia (TD) that can lead to lameness. A bead-based affinity matrix (ProteoMiner TM ) containing a combinatorial library of hexapeptides was used to deplete high abundant proteins and enrich the less abundant ones to compare between the sera of six-week old normal and TD affected chickens. Equal amounts of proteins in ProteoMiner depleted serum from control and TD-affected birds were subject to 2D gel electrophoresis, image analysis, and compared to identify the differentially expressed protein spots. The protein spots were characterized using in-gel trypsin digestion followed by mass spectrometry (MS). Of 46 matched protein spots in the gels, 33 were identified by peptide mass finger printing (PMF) and tandem mass spectrometry (MS/MS). Eight spots corresponding to immunoglobulins (Ig) were up-regulated in birds with TD and two spots down regulated. The up-regulated Ig proteins belonged to IgM and IgY(IgG) classes indicated by the identification of 'mu' chain and Fc fragment associated peptides respectively. Enzyme linked immunosorbent assay corroborated an increase in serum IgM levels but not IgG. Although the significance of the increase in IgM proteins in the serum of TD-affected chickens is not understood, it is likely that IgM plays some role in the removal of apoptotic chondrocytes which abound within TD lesions.
The objective of this study was to characterize and compare the water and salt soluble proteins from rat liver and muscle by using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and sodium dodecyl sulphate polymer-filled capillary gel electrophoresis (SDS-CGE). The evaluation and comparison of separations realized with the use of both techniques was done. It was found that although the main separation mechanism have similar in both cases. However, it was considered that, SDS-CGE seems to have a potential to be the alternative, efficient tool for the separation of tissue proteins based purely on their molecular weights.
Turmeric (Curcuma longa) has been shown to possess anti-inflammatory, antioxidant and antitumor properties. Extraction, partition and column chromatography of the dry powder of C. longa rhizomes showed presence of biological activity only in ethyl acetate eluted fraction in clonogenic assays using highly metastatic PC-3M prostate cancer cell line. HPLC, UV-Vis and Mass spectra studies showed presence of three curcuminoids in this fraction. Accordingly, we have made an attempt to identify the proteins modulated by purified turmeric fraction in PC-3M human prostate cancer cell line using high-resolution two-dimensional gel electrophoresis (2-DE). Isoelectric focusing and 2-DE analysis showed a total of 29 proteins altered by treatment with ethyl acetate fraction (EAF) of C. longa. Out of 29 differentially expressed pro- teins, 15 were identified by peptide mass fingerprinting through LC/MS/MS sequencing. From these, a total of 7 were down-regulated and 8 were up-regulated spots. The down-regulated proteins identified by Peptide Mass Fingerprinting are Elongation Factor 2 (eEF2), Stress-induced phosphoprotein 1, Glutathione S-transferase (GST) Omega-1, Parvalbumin alpha, Succinyl-CoA: ketoacid, Lamin-A/C and Annexin A2. The up-regulated proteins identified by Peptide Mass Fin- gerprinting include 78 kDa Glucose-regulated protein precursor (GRP78), Protein disulfide isomerase (PDI) precursor, Actin cytoplasmic 2, protein SET, Calreticulin precursor, Nucleophosmin, Vimentin, and Aortic alpha-actin (ACTA2). The identified proteins have diverse cellular functions such as ER stress, Unfolded Protein Response (UPR), cytoskeletal, structural, regulatory, and apoptotic proteins that will require further in-depth studies to understand the biological signifi- cance.
α-Synuclein (α-Syn) is a small intrinsically disordered presynaptic protein known to form insoluble filamentous inclusions in Parkinson's disease (PD) and other neurodegenerative disorders.Various catecholamines can inhibit the α-Syn fibrillation in vitro.Recently, non-covalent binding of DOPAC (3,4-dihydroxyphenylacetic acid), a normal product of the dopamine metabolism, was shown to inhibit the fibrillation of α-Syn due to the DOPAC-induced stabilization of the normally transient oligomers thus preventing them from subsequent fibril formation (Zhou, et al.J. Mol.Biol.2009, 388 (3), 597-610).We are showing here that the interaction of DOPAC with α-Syn decreases the binding affinity of α-Syn to lipids, suggesting that DOPAC might lead to the gain-of-toxicity of α-Syn aggregates and loss-of-function of α-Syn, both of which could be related to progression of PD.
The method for comparing bacterial proteomes has traditionally been two-dimensional gel electrophoresis (2-D GE); however, in recent years, new procedures for protein separation have been introduced.One of these new procedures utilizes column-based liquid chromatography (2-D LC) separation.The techniques by which these two methods separate proteins differ significantly; however, it is currently unclear to what degree the sets of proteins identified by these different methods will diverge.To address this question we compared the proteomes of Escherichia coli O157:H7 strain EDL933 against a naturally occurring variant using both 2-D GE and 2-D LC.Whole protein samples were prepared from the wild type and variant and split in half, with one half analyzed by 2-D GE and the other with the 2-D LC.Differentially regulated proteins were observed in each system and identified by MALD/I-TOF/TOF analysis.The differences in the protein detection sensitivities of Coomassie-blue stain used in 2-D GE and UV detectors used for 2-D LC resulted in different numbers of total proteins visualized in each system and therefore different numbers of matched protein pairs visualized during comparative assays.Despite the differences in visualization the numbers, but not the identities, of the differentially regulated proteins that could be identified by MALD/I analyses were similar for both 2-D GE and 2-D LC.However, a lack of significant redundancy between the sets of proteins identified suggests that these two methods are complimentary and not strictly corroborative.
The method for comparing bacterial proteomes has traditionally been two-dimensional gel electrophoresis (2-D GE); however, in recent years, new procedures for protein separation have been introduced. One of these new procedures utilizes column-based liquid chromatography (2-D LC) separation. The techniques by which these two methods separate proteins differ significantly; however, it is currently unclear to what degree the sets of proteins identified by these different methods will diverge. To address this question we compared the proteomes of Escherichia coli O157:H7 strain EDL933 against a naturally occurring variant using both 2-D GE and 2-D LC. Whole protein samples were prepared from the wild type and variant and split in half, with one half analyzed by 2-D GE and the other with the 2-D LC. Differentially regulated proteins were observed in each system and identified by MALD/I-TOF/TOF analysis. The differences in the protein detection sensitivities of Coomassie-blue stain used in 2-D GE and UV detectors used for 2-D LC resulted in different numbers of total proteins visualized in each system and therefore different numbers of matched protein pairs visualized during comparative assays. Despite the differences in visualization the numbers, but not the identities, of the differentially regulated proteins that could be identified by MALD/I analyses were similar for both 2-D GE and 2-D LC. However, a lack of significant redundancy between the sets of proteins identified suggests that these two methods are complimentary and not strictly corroborative.
Proteomics approaches enable interrogation of large numbers of proteins to provide a more comprehensive understanding of biological systems. High throughput proteomics typically utilizes liquid chromatography - mass spectrometry technology for data acquisition. Bioinformatic analysis tools are essential to manage and mine resulting high volume proteomics data sets. Data analysis is a current bottleneck for many proteomics researchers because complete and freely accessible already-developed systems are not available. In addition, most analysis systems require experienced bioinformatician input immediately upon data acquisition. For proteomics to achieve greatest impact in biology, data analysis must be more efficient and effective. We present the Proteome Discovery Pipeline (PDP), a web-based analysis platform that provides proteomics data analysis without requirement for specialized hardware or input from bioinformatics specialists for initial data analyses. Function- alities of the PDP include spectrum visualization, deconvolution, alignment, normalization, statistical significance tests, and pattern recognition. The PDP provides proteomic researchers with a user-friendly web-based data analysis package that can handle multiple file formats and facilitates data analysis from multiple proteomics technology platforms. The sys- tem is flexible and extensible to enable further development. In this paper the PDP development is described and the sys- tem capabilities are illustrated through a case study of human plasma proteomics data analysis.
Contrast Media is used in angiography, urography and tomography. It has been reported that contrast media nephrotoxicity incidence is 50% higher in patients with diabetic nephropathy undergoing coronary angiography and ap- proximately 15% of these patients developed renal failure. Proteomic analyses are a promising tool to study renal patho- physiology. In the future identification of biomarkers in renal diseases will develop therapeutic targets that will decrease the damage of acute renal failure. To identify proteins that were up or down-regulated in immortalized mesangial cells in a culture in the presence of contrast media we evaluated three groups: control, manitol used as control of osmolarity and Hexabrix TM . The cell homogenate from each group was submitted to the 2D-PAGE analysis, and proteins were identified by MALDI-TOF mass spectrometry. Some proteins were expressed only in the Hexabrix group (Proliferating Cell Nu- clear Antigen and Masp 1); others were up-regulated in the Manitol group (Translationally Controlled Tumor Protein), a protein was expressed only in the Control and Manitol (Annexin A3) groups, and we identified proteins that were ex- pressed in all three groups (Heat shock proteins 27 and 84, protein disulfide isomerase A3 precursor and Beta-actin 1). We believe that other substances present in Hexabrix, possibly the iodine, could be the regulatory factor of these proteins and the Hexabrix could be responsible for the nephrotoxicity observed in these cells.
A proteomics approach was used to study the proteins associated with chinch bug infested corn seedlings. Ex- amination of two-dimensional gels revealed the presence of more than 600 high quality protein spots each from chinch bug-infested and healthy corn seedlings. A total of 31 protein spots was selected for matrix-assisted laser desorption and ionization time-of-flight mass spectrometric analysis. Among the protein spots selected, 13 were from infested plants, 10 from healthy plants, and four each from healthy and infested plants having differential expressions. Peptide mass finger- printing revealed that each spot analyzed represents a different protein. Thirty-nine percent of the proteins had confirmed identity and the rest were tentatively identified. Among 13 proteins analyzed from infested seedlings most were related to defense, cell rescue, virulence and metabolism. Some of these proteins related to metabolism and protein synthesis were down-regulated in the infested seedlings. All proteins except one from infested corn seedlings seem to be activated in the plant system because of the chinch bug-induced stresses including osmotic, oxidative and acid stresses, and wounding.
Exiguobacterium sibiricum 255-15 has shown significantly improved cryotolerance after liquid broth growth at 4 o C and agar surface growth at both 4 o C and 25 o C compared with liquid broth growth at 25 o C. The ability to survive freeze-thaw stress is expected to depend on the physiological state and protein composition of cells prior to freezing.Using 2-D liquid separation and an ESI-TOF MS-based mass mapping technique, we examined the differences in the proteomic profiles of the permafrost bacterium E. sibiricum 255-15 grown at two temperatures (4 o C and 25 o C) and two media (liquid broth and agar surface) before freeze-thawing treatments.In this study, a total of 330 proteins were identified.The cells cultured under the growth conditions associated with the improved cryotolerance have revealed a general downregulation of enzymes involved in major metabolic processes (glycolysis, anaerobic respiration, ATP synthesis, fermentation, electron transport, and sugar metabolism) as well as in the metabolism of lipids, amino acids, nucleotides and nucleic acids.In addition, eight proteins (2'-5' RNA ligase, hypoxanthine phosphoribosyl transferase, FeS assembly ATPase SufC, thioredoxin reductase and four hypothetical proteins) were observed to be up-regulated.This suggests these eight proteins might have a potential role to induce the improved cryotolerance.
There are few reports describing the role of p21-dependent protein repression in cell death.To identify such cell death-associated proteins and to shed the light into the molecular mechanisms by which p21 is responding to pharmacological stress, we used a subcellular proteomic approach for the analysis of protein expression profiles of fractionated nuclei, mitochondria, and cytosols of isogenic p21 null (p21-/-) and wild-type human HCT-116 cells following treatment with sublethal doses (1μM) of the topoisomerase I inhibitor, topotecan (TPT).In total, 174 unique deregulated proteins were identified in HCT-116 cells following treatment with TPT, whereas only 146 proteins were identified in p21-/-cells.They contributed to multiple functional activities of stress signaling pathways, and that p21-/-cells are accelerated to be more responsive to topotecan-induced cell death due to the following: 1) down regulation of proteins involved in the transcriptional and replication machinery of cells like DNA (cytosine-5)-methyltransferase 1, Matrin 3, DNA replication licensing factor MCM4, heterogeneous nuclear ribonucleoprotein Q, poly(Rc)-binding protein 1 and splicing factor arginine serine rich 7; 2) the activation of a caspase-independent apoptosis by the upregulation of the Bcl2 inhibitor of transcription (Bit1) protein; and 3) the activation of TNF signaling by the upregulation of macrophage immigration inhibitory factor (MIF), and 26S proteosome non-ATPase regulatory subunit 2 (TRAP2) proteins.We suggest that the upregulation of these proteins are contributing factors to the molecular mechanisms of topotecan-induced cell death in p21-/-cells; and that the data present an opportunity for developing new therapeutic approaches for selective targeting of p21signaling pathways.
The microenvironment of the central nervous system is important for neuronal function and development. Dur- ing the early stages of embryo development the cephalic vesicles are filled by embryonic cerebrospinal fluid, a complex fluid containing different protein fractions, which contributes to the regulation of the survival, proliferation and neuro- genesis of neuroectodermal stem cells. The protein content of embryonic cerebrospinal fluid from chick and rat embryos at the start of neurogenesis has already been determined. Most of the identified gene products are thought to be involved in the regulation of developmental processes during embryogenesis. However, due to the crucial roles played by embry- onic cerebrospinal fluid during brain development, the embryological origin of the gene products it contains remains an intriguing question. According to the literature most of these products are synthesised in embryonic tissues other than the neuroepithelium. In this study we examined the embryological origin of the most abundant embryonic cerebrospinal fluid protein fractions by means of slot-blot analysis and by using several different embryonic and extraembryonic protein ex- tracts, immunodetected with polyclonal antibodies. This first attempt to elucidate their origin is not based on the proteins identified by proteomic methods, but rather on crude protein fractions detected by SDS-PAGE analysis and to which polyclonal antibodies were specifically generated. Despite some of the limitations of this study, i.e. that one protein frac- tion may contain more than one gene product, and that a specific gene product may be contained in different protein frac- tions depending on post-translational modifications, our results show that most of the analysed protein fractions are not produced by the cephalic neuroectoderm but are rather stored in the egg reservoir; furthermore, few are produced by em- bryo tissues, thus indicating that they must be transported from their production or storage sites to the cephalic cavities, most probably via embryonic serum. These results raise the question as to whether the transfer of proteins from these two embryo compartments is regulated at this early developmental stage.
Candida albicans is the most commonly isolated fungus associated with biofilms, which are extracellular ma- trix (ECM)-encased, drug-resistant microbial communities formed on indwelling medical devices. Protein profiles of fun- gal biofilms have not been investigated in detail, although such profiles are believed to play critical roles in fungal biofilm formation. In this study, we used two-dimensional difference-in-gel electrophoresis (DIGE)-based proteomics to identify differentially expressed proteins in C. albicans biofilms grown to early and mature phases, compared to planktonic cells. The resulting proteomic data set was subjected to pathway mapping to reveal phase-specific pathways that were differen- tially expressed in biofilm cell walls and extracellular matrix (ECM). Our analyses showed 107 proteins to be differen- tially expressed in ECM, while 44 were differentially expressed in cell walls during biofilm formation, compared to planktonic controls. Furthermore, 95% (102/107) and 68% (30/44) of these differentially expressed proteins were upregu- lated in ECM and cell walls of biofilms, respectively. These proteins were mapped to cellular pathways, which revealed that these differentially expressed proteins were associated with several metabolic pathways, in a phase-dependent man- ner. For example, among ECM-associated proteins, proteins within 18 pathways were differentially expressed, with two pathways (glutamate and nitrogen metabolism) unique to early phase, and four pathways (purine, Gly/Ser/Thr, and inosi- tol metabolism, and carbon fixation) unique to mature phase biofilms. Such differences were also observed in cell wall- associated proteins, where proteins associated with 14 specific pathways were differentially regulated. We also found gly- colytic enzymes including the key enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were overexpressed in biofilms at both early and mature phases, compared to planktonic controls. Iodoacetate-mediated inhibition of this enzyme completely abrogated the ability of C. albicans to form biofilms, indicating the role of glycolysis/gluconeogenesis path- ways in biofilm formation. Taken together, we demonstrate that ECM and cell walls of C. albicans biofilms express in- creased levels of specific proteins within pathways in a phase-dependent manner, suggesting that these pathways, espe- cially glycolysis/gluconeogenesis, play critical roles in fungal biofilm formation and maintenance.
By using a differential proteomic approach, responses to drought stress in sunflower have been studied. Two sunflower genotypes, showing different levels of tolerance to drought have been utilized. Following TCA-acetone protein extraction, the 2-DE leaf protein profile of well watered and drought stressed plants have been compared. Coomassie staining of the gels allowed visualization of around 350 well resolved spots within the 5-8 pH and 10-100 kDa ranges. Image analysis revealed the presence of both, qualitative and quantitative changes between genotypes and treatments. Dif- ferential spots were subjected to trypsin digestion and peptides were analyzed by MALDI-TOF mass spectrometry. After database search using peptide mass fingerprinting, 2 genotype-dependent and 23 (susceptible genotype) and 5 (tolerant genotype) stress-responsive protein spots were identified. The two proteins spots differentiating sunflower genotypes cor- responded to phosphoglycerate kinase and glyceraldehyde-3-phosphate dehydrogenase. In response to drought conditions a general decrease in protein spots corresponding to enzymes of the photosynthesis and carbohydrate metabolism was ob- served in the more susceptible genotype, suggesting inhibition of the energetic metabolism. Such changes have not been observed in the tolerant genotype, indicating a normal metabolism under drought stress.
Tryptophan plays an important role in vertebrate metabolism as not only a building block of proteins, but also as a precursor of serotonin, melatonin, niacin and kynurenines, which influence immune tolerance.Here we use an animal paradigm and quantitative serum proteomics to model tryptophan deficiency.We applied bidirectional H 2 16/18 O labeling to serum proteins from chickens fed either a tryptophan-deficient or-adequate diet and used the plant protein RuBisCO as an internal standard.The proteins were trypsin digested and processed by 2-dimensional liquid chromatography electrospray ionization tandem mass spectrometry (2D LC ESI MS 2 ).The resulting mass spectra were analyzed using the SEQUEST algorithm and the ProteinMapper program to identify proteins that had increased or decreased expression.We identified 4161 proteins labeled bidirectionally, of which 46 were increased and 90 decreased (~3%).Using Ingenuity Pathways Analysis (IPA) software, we found that a tryptophan nutritional deficiency may affect not only the immune and neurological systems, but our modeling also suggests that it may be important in cancer, optic atrophy and cardiomyopathy.
