Proteomic profiling of Cronobacter turicensis 3032, a food‐borne opportunistic pathogen

Members of the genus Cronobacter are opportunistic pathogens for neonates and are often associated with contaminated milk powder formulas. At present little is known about the virulence mechanisms or the natural reservoir of these organisms. The proteome of Cronobacter turicensis 3032, which has recently caused two deaths, was mapped aiming at a better understanding of physiology and putative pathogenic traits of this clinical isolate. Our analyses of extracellular, surface‐associated and whole‐cell proteins by two complementary proteomics approaches, 1D‐SDS‐PAGE combined with LC‐ESI‐MS/MS and 2D‐LC‐MALDI‐TOF/TOF MS, lead to the identification of 832 proteins corresponding to a remarkable 19% of the theoretically expressed protein complement of C. turicensis. The majority of the identified proteins are involved in central metabolic pathways, translation, protein folding and stability. Several putative virulence factors, whose expressions were confirmed by phenotypic assays, could be identified: a macrophage infectivity potentiator involved in C. turicensis persistence in host cells, a superoxide dismutase protecting the pathogen against reactive oxygen species and an enterobactin‐receptor protein for the uptake of siderophore‐bound iron. Most interestingly, a chitinase and a metalloprotease that might act against insects and fungi but no casein hydrolysing enzymes were found, suggesting that there is an environmental natural habitat of C. turicensis 3032.     

Proteomic changes in hearts of kyphoscoliosis (ky) mutant mice in the absence of structural pathology: implication for the analysis of early human heart disease

Complex molecular changes associated with early stage human heart disease are poorly understood and prevent the development of effective treatments of human cardiac disease. Relatively minor structural changes in early disease may accompany some conditions such as arrhythmias. Our objective was to determine if significant proteomic changes occur in heart tissues in the absence of structural pathology. We used a proteomic "pipeline" based on Ciphergen SELDI-TOF/MS, gel electrophoresis and MALDI-TOF/MS. The kyphoscoliosis (ky) mouse carries a mutation in a putative transglutaminase causing a primary skeletal muscle disease. The ky protein is expressed usually in skeletal and cardiac muscle but its absence from the ky heart causes no structural pathology making it a good model of "occult" heart disease. We discovered 20 statistically validated biomarkers discriminating ky from normal hearts, one cardiac troponin-I was reduced by 40% in ky hearts. A 17% deficit was confirmed subsequently by Western blot. Thus, the proteome of ky hearts was abnormal, giving support to our contention that this SELDI-based analytical approach is capable of making a significant contribution to the analysis of complex proteomic changes in early stage human heart disease.     

Proteomic analysis of inflammatory biomarkers in bronchoalveolar lavage

To assess markers of lung inflammation, we used SELDI-TOF and 2-DE to study changes in bronchoalveolar lavage (BAL) protein in 33 subjects challenged with local bronchial lung endotoxin and saline and in 11 patients with acute respiratory distress syndrome (ARDS). Differences in the SELDI-TOF spectra were assessed by t-test after baseline subtraction, normalization to total ion current and alignment by m/z calibration. The temporal changes in acute inflammatory BAL (6, 24 and 48 h following endotoxin challenge) on hydrophobic binding chip surfaces revealed the differential presence of proteins of 9, 14, 18 and 28 kDa (all p <0.001) in the inflammatory BAL. This differential pattern was also found in the ARDS BAL. Principal component analysis of the entire SELDI-TOF spectra separated normal BAL, experimental and clinical lung inflammation supporting the notion of a distinctive protein pattern associated with acute lung inflammation. An analysis of the hydrophobic fraction of the inflammatory BAL using 2-DE, identified increased levels of apolipoprotein A1, and S100 calcium-binding proteins A8 and A9 in the inflammatory BAL. This pattern was also found in ARDS BAL after immunoblot analysis. These approaches will be useful to improve current methods of monitoring lung inflammation and to identify new therapeutic targets.     

Proteomic analysis of colonic mucosa in a rat model of irritable bowel syndrome

Irritable bowel syndrome (IBS) is one of the most common functional disorders of the gastrointestinal tract. It is characterized by abdominal pain and changes in bowel habits. Various studies have investigated the pathophysiologic processes underlying IBS, but the mechanism remains poorly understood. In the present study, we established an IBS model and identified differentially expressed proteins in colon tissue of IBS rats compared with healthy controls by 2‐D gel electrophoresis, MALDI‐TOF‐MS, and Western blot analysis. Our results showed that 13 of the 1396 protein spots on 2‐D gel were differently expressed between the IBS and control groups. Ontological analysis of these proteins revealed primary roles in catalytic activity (protein disulfide‐isomerase A3, glyoxalase I, cathepsin S, α‐enolase), structural support (cytokeratin 8), antioxidant activity (peroxiredoxin‐6), protein binding (transgelin, serpin peptidase inhibitor B5), and signal transduction (40S ribosomal protein SA). Protein disulfide‐isomerase A3 and cytokeratin 8 overexpression in IBS were confirmed by Western blot. The findings indicate that multiple proteins are involved in IBS processes that influence intestinal tract immunity, inflammation, and nerve regulation. Our study provides useful candidate genes and proteins for further investigation.     

Predicting left ventricular remodeling after a first myocardial infarction by plasma proteome analysis

Recent improvements in therapeutic strategies did not prevent left ventricular remodeling (LVR), which remains a common event (30%) after acute myocardial infarction (AMI). We report the use of a systematic approach, based on comparative proteomics, to select circulating biomarkers that may be associated with LVR. We selected 93 patients enrolled in a prospective study. These patients with anterior wall Q-wave AMI underwent echocardiographic follow-up at hospitalization, 3 months and 1 year after AMI. They were divided into three groups (no, low, or high remodeling). Plasma samples of these patients (day 5 of hospitalization) were processed and stored at -80 degrees C within 2 h and analyzed using SELDI-TOF protein chip technology. This systematic approach allowed to select candidate proteins modulated by LVR: post-translational variants of alpha1-chain of haptoglobin (Hpalpha1) corresponding to m/z 9493, 9565, and 9623, which were more elevated in remodeling patients. The peak 9493 m/z was shown having a receiving-operating characteristic (ROC) value of 0.71 between non- and remodeling patients. SELDI-TOF approach may lead to the identification of circulating proteins associated with LVR. Whether these candidate proteins will help to identify patients who are at high risk of heart failure after AMI will have to be tested in future studies.     

Proteomic analysis of the cold stress response in the moss,Physcomitrella patens

Cold stress has adverse effects on plant growth and development. Plants respond and acclimate to cold stress through various biochemical and physiological processes, thereby acquiring stress tolerance. To better understand the basis for tolerance, we carried out a proteomic study in the model moss, Physcomitrella patens, characterizing gametophore proteins with 2‐DE and mass spectroscopy. Following exposure to 0°C for up to 3 days, out of the more than 1000 protein spots reproducibly resolved, only 45 changed in abundance by at least 1.5‐fold. Of these, 35 were identified by tryptic digestion and mass spectroscopy. Photosynthetic proteins decreased, whereas many catabolic proteins increased. In addition, cold stress up‐regulated a variety of signaling, cytoskeleton, and defense proteins and few proteins in these classes were down‐regulated. Up‐regulated proteins include the 14‐3‐3‐like protein, actin, HSP70s, lipoxygenases, and cytochrome P450 proteins. These results point to pathways that are important for the mechanism of cold stress response in P. patens and by extension to the entire plant kingdom.     

Proteomic analysis of the dorsal and ventral hippocampus of rats maintained on a high fat and refined sugar diet

The typical Western diet, rich in high saturated fat and refined sugar (HFS), has been shown to increase cognitive decline with aging and Alzheimer's disease, and to affect cognitive functions that are dependent on the hippocampus, including memory processes and reversal learning. To investigate neurophysiological changes underlying these impairments, we employed a proteomic approach to identify differentially expressed proteins in the rat dorsal and ventral hippocampus following maintenance on an HFS diet. Rats maintained on the HFS diet for 8 weeks were impaired on a novel object recognition task that assesses memory and on a Morris Water Maze task assessing reversal learning. Quantitative label‐free shotgun proteomic analysis was conducted on biological triplicates for each group. For the dorsal hippocampus, 59 proteins were upregulated and 36 downregulated in the HFS group compared to controls. Pathway ana‐lysis revealed changes to proteins involved in molecular transport and cellular and molecular signaling, and changes to signaling pathways including calcium signaling, citrate cycle, and oxidative phosphorylation. For the ventral hippocampus, 25 proteins were upregulated and 27 downregulated in HFS fed rats. Differentially expressed proteins were involved in cell‐to‐cell signaling and interaction, and cellular and molecular function. Changes to signaling pathways included protein ubiquitination, ubiquinone biosynthesis, oxidative phosphorylation, and mitochondrial dysfunction. This is the first shotgun proteomics study to examine protein changes in the hippocampus following long‐term consumption of a HFS diet, identifying changes to a large number of proteins including those involved in synaptic plasticity and energy metabolism. All MS data have been deposited in the ProteomeXchange with identifier PXD000028.     

Proteomic analysis of human papillomavirus‐related oral squamous cell carcinoma: Identification of thioredoxin and epidermal‐fatty acid binding protein as upregulated protein markers in microdissected tumor tissue

Human papillomavirus (HPV) infection has been identified as an etiologic agent for a subset of oral squamous cell carcinoma (OSCC) with increasing incidence. HPV DNA‐positivity may confer better prognosis but the related oncogenic mechanisms are unknown. For the identification of HPV relevant proteins, we analyzed microdissected cells from HPV DNA‐positive (n = 17) and HPV DNA‐negative (n = 7) OSCC tissue samples. We identified 18 proteins from tumor tissues by peptide fingerprint mapping and SELDI MS that were separated using 2‐DE. Among a number of signals that were detected as significantly different in the protein profiling analysis, we identified thioredoxin (TRX) and epidermal‐fatty acid binding protein as upregulated in HPV related tumor tissue. This study, investigating for the first time proteomic changes in microdissected HPV infected tumor tissue, provides an indication on the oncogenic potential of viruses.     

Comparative proteomic analysis of human lung telocytes with fibroblasts

Telocytes (TCs) were recently described as interstitial cells with very long prolongations named telopodes (Tps; www.telocytes.com ). Establishing the TC proteome is a priority to show that TCs are a distinct type of cells. Therefore, we examined the molecular aspects of lung TCs by comparison with fibroblasts (FBs). Proteins extracted from primary cultures of these cells were analysed by automated 2‐dimensional nano‐electrospray ionization liquid chromatography tandem mass spectrometry (2D Nano‐ESI LC‐MS/MS). Differentially expressed proteins were screened by two‐sample t‐test (P < 0.05) and fold change (>2), based on the bioinformatics analysis. We identified hundreds of proteins up‐ or down‐regulated, respectively, in TCs as compared with FBs. TC proteins with known identities are localized in the cytoskeleton (87%) and plasma membrane (13%), while FB up‐regulated proteins are in the cytoskeleton (75%) and destined to extracellular matrix (25%). These identified proteins were classified into different categories based on their molecular functions and biological processes. While the proteins identified in TCs are mainly involved in catalytic activity (43%) and as structural molecular activity (25%), the proteins in FBs are involved in catalytic activity (24%) and in structural molecular activity, particularly synthesis of collagen and other extracellular matrix components (25%). Anyway, our data show that TCs are completely different from FBs. In conclusion, we report here the first extensive identification of proteins from TCs using a quantitative proteomics approach. Protein expression profile shows many up‐regulated proteins e.g. myosin‐14, periplakin, suggesting that TCs might play specific roles in mechanical sensing and mechanochemical conversion task, tissue homoeostasis and remodelling/renewal. Furthermore, up‐regulated proteins matching those found in extracellular vesicles emphasize TCs roles in intercellular signalling and stem cell niche modulation. The novel proteins identified in TCs will be an important resource for further proteomic research and it will possibly allow biomarker identification for TCs. It also creates the premises for understanding the pathogenesis of some lung diseases involving TCs.     

Comparative proteomics profile of osteoblasts cultured on dissimilar hydroxyapatite biomaterials: an iTRAQ-coupled 2-D LC-MS/MS analysis

Hydroxyapatite (HA) and its derived bioceramic materials have been widely used for skeletal implants and/or bone repair scaffolds. It has been reported that carbon nanotube (CNT) is able to enhance the brittle ceramic matrix without detrimental to the bioactivity. However, interaction between osteoblasts and these bioceramics, as well as the underlying mechanism of osteoblast proliferation on these bioceramic surfaces remain to be determined. Using iTRAQ-coupled 2-D LC-MS/MS analysis, we report the first comparative proteomics profiling of human osteoblast cells cultured on plane HA and CNT reinforced HA, respectively. Cytoskeletal proteins, metabolic enzymes, signaling, and cell growth proteins previous associated with cell adhesion and proliferation were found to be differentially expressed on these two surfaces. The level of these proteins was generally higher in cells adhered to HA surface, indicating a higher level of cellular proliferation in these cells. The significance of these findings was further assessed by Western blot analysis. The differential protein profile in HA and CNT strengthened HA established in our study should be valuable for future design of biocompatible ceramics.     

Proteomic analysis of Arabidopsis thaliana (L.) Heynh responses to a generalist sucking pest (Myzus persicae Sulzer)

Herbivorous insects can cause severe cellular changes to plant foliage following infestations, depending on feeding behaviour. Here, a proteomic study was conducted to investigate the influence of green peach aphid (Myzus persicae Sulzer) as a polyphagous pest on the defence response of Arabidopsis thaliana (L.) Heynh after aphid colony establishment on the host plant (3 days). Analysis of about 574 protein spots on 2‐DE gels revealed 31 differentially expressed protein spots. Twenty out of these 31 differential proteins were selected for analysis by mass spectrometry. In 12 of the 20 analysed spots, we identified seven and nine proteins using MALDI‐TOF‐MS and LC‐ESI‐MS/MS, respectively. Of the analysed spots, 25% contain two proteins. Different metabolic pathways were modulated in Arabidopsis leaves according to aphid feeding: most corresponded to carbohydrate, amino acid and energy metabolism, photosynthesis, defence response and translation. This paper has established a survey of early alterations induced in the proteome of Arabidopsis by M. persicae aphids. It provides valuable insights into the complex responses of plants to biological stress, particularly for herbivorous insects with sucking feeding behaviour.     

A comparative proteomic analysis of HepG2 cells incubated by S(−) and R(+) enantiomers of anti‐coagulating drug warfarin

Warfarin is a commonly prescribed oral anti‐coagulant with narrow therapeutic index. It interferes with vitamin K cycle to achieve anti‐coagulating effects. Warfarin has two enantiomers, S(?) and R(+) and undergoes stereoselective metabolism, with the S(?) enantiomer being more effective. We reported that the intracellular protein profile in HepG2 cells incubated with S(?) and R(+) warfarin, using iTRAQ‐coupled 2‐D LC‐MS/MS. In samples incubated with S(?) and R(+) warfarin alone, the multi‐task protein Protein SET showed significant elevation in cells incubated with S(?) warfarin but not in those incubated with R(+) warfarin. In cells incubated with individual enantiomers of warfarin in the presence of vitamin K, protein disulfide isomerase A3 which is known as a glucose‐regulated protein, in cells incubated with S(?) warfarin was found to be down‐regulated compared to those incubated with R(+) warfarin. In addition, Protein DJ‐1 and 14‐3‐3 Proteinσ were down‐regulated in cells incubated with either S(?) or R(+) warfarin regardless of the presence of vitamin K. Our results indicated that Protein DJ‐1 may act as an enzyme for expression of essential enzymes in vitamin K cycle. Taken together, our findings provided molecular evidence on a comprehensive protein profile on warfarin–cell interaction, which may shed new lights on future improvement of warfarin therapy.     

Proteomic analysis of anaplastic lymphoma cell lines: identification of potential tumour markers

Anaplastic large-cell lymphomas (ALCL) are high grade lymphomas of T or null phenotype often associated with the t(2;5) translocation leading to the expression of a chimeric protein consisting of the N-terminal portion of nucleophosmin (NPM) and the intracellular domain of the anaplastic lymphoma kinase (ALK). Although ALCL are recognized as distinct clinical, biological and cytogenetic entities, heterogeneities persist in this group of tumours, which exhibit a broad spectrum of morphological features. Particularly, the common type tumour consisting in large cells contrast with the small cell variant that is sometimes associated with a leukemic phase. The ALK-negative ALCL is often associated with a poor prognosis. Here, we investigated the proteome of these subtypes of tumours using patient-derived cell lines. We compared the proteome of the cytosolic fraction of NPM-ALK-positive versus NPM-ALK-negative cells on one hand, and the proteome of common cell type versus small cell variant on the other hand. The identification of a set of proteins differentially expressed in the subtypes of ALCL points to new diagnosis/prognosis markers. This study also provides interesting information on the molecular mechanisms responsible for the different subtypes of ALCL.     

Proteomic analysis of the response of an acidophilic strain of Chlamydomonas sp. (Chlorophyta) to natural metal‐rich water

A proteomic approach including 2‐DE and MALDI‐TOF analysis has been developed to identify the soluble proteins of the unicellular photosynthetic algae Chlamydomonas sp. isolated from an extreme acidic environment, Río Tinto (southwest Spain). We have analyzed the soluble proteome obtained from whole cells growing on metal‐rich natural acidic water from the river in comparison with the same strain growing in artificial BG‐11 media. The most drastic effect was the decrease in the abundance of the ribulose‐1,5‐biphosphate carboxylase as well as other enzymes related to photosynthesis. However, phytochrome B, phosphoribulokinase, and phosphoglycerate kinase were upregulated when cells were grown in metal‐rich acidic water. Besides, increased accumulation of two Hsps, Hsp70 and Hsp90 as well as other stress‐related enzymes were also found in the cells growing in natural acidic water. These results suggest that naturally occurring metal‐rich water induces a stress response in acidophilic Chlamydomonas forcing algal cells to reorganize their metabolic pathways as an adaptive response to these environmental conditions.     

Two‐dimensional proteome reference map of Prototheca zopfii revealed reduced metabolism and enhanced signal transduction as adaptation to an infectious life style

Biochemical, serological, and genetic analyses have identified two genotypes of Prototheca zopfii, a unicellular microalga belonging to the family Chlorellaceae. The P. zopfii genotype 1, abundantly present in cow barns and environment, remains nonpathogenic, while P. zopfii genotype 2 has been isolated from cows with bovine mastitis. The present study was carried out to identify the protein expression level difference between the pathogenic and nonpathogenic strains of P. zopfii. A total of 782 protein spots were observed on the 2D fluorescence difference gel electrophoresis (2D DIGE) gels among which 63 and 44 proteins were identified to be overexpressed in genotypes 1 and 2, respectively. The limited number of protein entries specific for Prototheca in public repositories resulted mainly in the identification of proteins described in other algae, microorganisms, or plants. Gene ontology (GO) analysis indicated reduced carbohydrate metabolism in genotype 1, while genotype 2 displayed enhanced DNA binding, kinase activity, and signal transduction. These effects point to metabolic and signaling adaptations in the pathogenic strain and provide insights into the evolution of otherwise highly similar strains. All MS data have been deposited in the ProteomeXchange with identifier PXD000126.     

Pre‐fractionation of rat liver cytosol proteins prior to mass spectrometry‐based proteomic analysis using tandem biomimetic affinity chromatography

Efficient and high resolution separation of the protein mixture prior to trypsin digestion and mass spectrometry (MS) analysis is generally used to reduce the complexity of samples, an approach that highly increases the probability of detecting low‐copy‐number proteins. Our laboratory has constructed an affinity ligand library composed of thousands of ligands with different protein absorbance effects. Structural differences between these ligands result in different non‐bonded protein–ligand interactions, thus each ligand exhibits a specific affinity to some protein groups. In this work, we first selected out several synthetic affinity ligands showing large band distribution differences in proteins absorbance profiles, and a tandem composition of these affinity ligands was used to distribute complex rat liver cytosol into simple subgroups. Ultimately, all the fractions collected from tandem affinity pre‐fractionation were digested and then analyzed by LC‐MS/MS, which resulted in high confidence identification of 665 unique rat protein groups, 1.8 times as many proteins as were detected in the un‐fractionated sample (371 protein groups). Of these, 375 new proteins were identified in tandem fractions, and most of the proteins identified in un‐fractionated sample (290, 80%) also emerged in tandem fractions. Most importantly, 430 unique proteins (64.7%) only characterized in specific fractions, indicating that the crude tissue extract was well distributed by tandem affinity fractionation. All detected proteins were bioinformatically annotated according to their physicochemical characteristics (such as MW, pI, GRAVY value, TM Helices). This approach highlighted the sensitivity of this method to a wide variety of protein classes. Combined usage of tandem affinity pre‐fractionation with MS‐based proteomic analysis is simple, low‐cost, and effective, providing the prospect of broad application in proteomics. Copyright © 2009 John Wiley & Sons, Ltd.     

Proteomic analysis of differential protein expression in response to epidermal growth factor in neonatal porcine pancreatic cell monolayers

We have proposed that porcine neonatal pancreatic cell clusters (NPCCs) may be a useful alternative source of cells for islet transplantation, and that monolayer cultures might provide an opportunity to manipulate the cells before transplantation. In addition we previously identified 10 genes up-regulated by epidermal growth factor (EGF) in cultured porcine NPCC monolayers. We have now analyzed the intracellular signaling pathways activated by EGF and searched for proteins differentially expressed following EGF treatment of the monolayers, using two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). EGF treatment resulted in phosphorylation of both Erk 1/2 and Akt, as well as increased cell proliferation. Five unknown and 13 previously identified proteins were differentially expressed in response to EGF. EGF treatment increased the expression of several structural proteins of epithelial cells, such as cytokeratin 19 and plakoglobin, whereas vimentin, the intermediate filament protein of mesenchymal cells, and non-muscle myosin alkali chain isoform 1, decreased. Heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1 factor, which promotes epithelial cell proliferation, and hemoglobin alpha I & II also increased, whereas cyclin A1, immunoglobulin heavy chain, apolipoprotein A1, 5,10-ethylenetetrahydrofolated reductase (5,10-MTHFR), angiotensin-converting enzyme 2 (ACE2), co-lipase II precursor, and NAD+ isocitrate dehydrogenase (NAD+ IDH) alpha chain proteins decreased. Our results show that EGF stimulates proliferation of pancreatic epithelial cells by simultaneously activating the MAPK and PI-3K pathways. HnRNP A2/B1, hemoglobin, cyclin A1, and ACE2 may play roles in the proliferation of epithelial cells in response to EGF.     

Enantioselective analysis of citalopram and demethylcitalopram in human and rat plasma by chiral LC-MS/MS: application to pharmacokinetics

Citalopram (CITA) is available as a racemic mixture and as a pure enantiomer. Its antidepressive action is related to the (+)-(S)-CITA and to the metabolite (+)-(S)-demethylcitalopram (DCITA). In the present investigation, a method for the analysis of CITA and DCITA enantiomers in human and rat plasma was developed and applied to the study of pharmacokinetics. Plasma samples (1 ml) were extracted at pH 9.0 with toluene:isoamyl alcohol (9:1, v/v). The CITA and DCITA enantiomers were analyzed by LC-MS/MS on a Chiralcel OD-R column. Recovery was higher than 70% for both enantiomers. The quantification limit was 0.1 ng/ml, and linearity was observed up to 500 ng/ml plasma for each CITA and DCITA enantiomer. The method was applied to the study of the kinetic disposition of CITA administered in a single oral dose of 20 mg to a healthy volunteer and in a single dose of 20 mg/kg (by gavage) to Wistar rats (n = 6 for each time). The results showed a higher proportion of the (-)-(R)-CITA in human and rat plasma, with S/R AUC ratios for CITA of 0.28 and 0.44, respectively. S/R AUC ratios of DCITA were 0.48 for rats and 1.04 for the healthy volunteer.