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PubMed2006

Comparative proteomic profiles of meningioma subtypes.

Meningiomas are classified into three groups (benign, atypical, and anaplastic) based on morphologic characteristics. Atypical meningiomas, which are WHO grade 2 tumors, and anaplastic meningiomas, which are WHO grade 3 tumors, exhibit an increased risk of recurrence and premature death compared with benign WHO grade 1 tumors. Although atypical and anaplastic meningiomas account for <10% of all of meningiomas, it can be difficult to distinguish them from benign meningiomas by morphologic criteria alone. We used selective tissue microdissection to examine 24 human meningiomas and did two-dimensional gel electrophoresis to determine protein expression patterns. Proteins expressed differentially by meningiomas of each WHO grade were identified and sequenced. Proteomic analysis revealed protein expression patterns unique to WHO grade 1, 2, and 3 meningiomas and identified 24 proteins that distinguish each subtype. Fifteen proteins showed significant changes in expression level between benign and atypical meningiomas, whereas nine distinguished atypical from anaplastic meningiomas. Differential protein expression was confirmed by Western blotting and immunohistochemistry. We established differential proteomic profiles that characterize and distinguish meningiomas of increasing grades. The proteins and proteomic profiles enhance understanding of the pathogenesis of meningiomas and have implications for diagnosis, prognosis, and treatment.

Electrophoresis, Gel, Two-Dimensional↗
PubMed2001

Comparative proteomics of bacterial pathogens.

The monitoring of gene expression via the technologies encompassed under the term 'proteomics' allows proteins of significance to be related to phenotypes associated with strain variability, environmental influences and the effects of genetic manipulation. The characterizations of these molecules are routinely performed utilising two-dimensional (2-D) gel electrophoresis in association with mass spectrometry for the identification of proteins. Pathogenic bacteria are suitable for proteomic comparisons in the aim of elucidating proteins with vaccine and diagnostic applications, as well as determining novel targets for drug design and the effects of these drugs on cellular physiology. Strains exhibiting diverse phenotypes including antibiotic or chemical resistances, altered mode of pathogenicity, or differential capability of growth in similar environments, can be compared via protein differential display to correlate relative protein abundances associated with these conditions. Technically, proteins are 'mapped' on 2-D arrays under 'standard' conditions and visually compared to arrays of proteins from a variety of test conditions. High-throughput technologies allow molecules of significance to be elucidated rapidly from within complex mixtures using a combination of cellular pre fractionation to determine cellular location and pathway predictions to aid in overcoming the limitations of 2-D gel technology for the analysis of whole proteomes.

Bacteria↗
PubMed2003

The comparative proteomics of ubiquitination in mouse.

Ubiquitination is a common posttranslational modification in eukaryotic cells, influencing many fundamental cellular processes. Defects in ubiquitination and the processes it mediates are involved in many human disease states. The ubiquitination of a substrate involves four classes of enzymes:a ubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzyme (E2), a ubiquitin protein ligase (E3), and a de-ubiquitinating enzyme (DUB). A substantial number of E1s (four), E2s (13), E3s (97), and DUBs (six) that were previously unknown in the mouse are included in the FANTOM2 Representative Transcript and Protein Set (RTPS). Many of the genes encoding these proteins will constitute promising candidates for involvement in disease. In addition, the RTPS provides the basis for the most comprehensive survey of ubiquitination-associated proteins across eukaryotes undertaken to date. Comparisons of these proteins across human and other organisms suggest that eukaryotic evolution has been associated with an increase in the number and diversity of E3s (possessing either zinc-finger RING, F-box, or HECT domains) and DUBs (containing the ubiquitin thiolesterase family 2 domain). These increases in numbers are too large to be accounted for by the presence of fragmentary proteins in the data sets examined. Much of this innovation appears to have been associated with the emergence of multicellular organisms, and subsequently of vertebrates, increasing the opportunity for complex regulation of ubiquitination-mediated cellular and developmental processes.

Animals↗
PubMed2001

Comparative proteome analysis of Leishmania donovani at different stages of transformation from promastigotes to amastigotes.

To pass through its life cycle, protozoan parasites of the genus Leishmania have to differentiate from promastigotes to amastigotes. The molecular basis underiving this major transformation is poorly understood. One way to study this phenomenon is to isolate and characterize proteins that are specifically expressed in one of the two stages of the life cycle or during the stage differentiation. Using two-dimensional gel electrophoresis, we mapped the Leishmania donovani proteome during stage differentiation to identify stage-specific proteins and regulons. A protocol for extracting proteins of both promastigote and amastigote L. donovani cells was developed, which is compatible with isoelectric focusing. Up to 400 L. donovani protein spots were visualized on a silver-stained gel. Metabolic labeling of the cells was used to compare directly the protein synthesis pattern with the protein level pattern. The silver-stained images of L. donovani cells harvested on different days of stage differentiation were compared to the corresponding autoradiographs. A marked decrease in protein synthesis during stage differentiation from promastigotes to amastigotes was observed. The stained protein pattern as well as the protein pattern on the autoradiograph changed dramatically, especially after day 3 (about 24 h after pH shift) of transformation.

Animals↗
PubMed2003

Comparative proteome analysis of culture supernatant proteins of Mycobacterium tuberculosis H37Rv and H37Ra.

To examine the virulence factors of Mycobacterium tuberculosis H37Rv, the proteome was used to characterize the differences in protein expression between virulent M. tuberculosis H37Rv and attenuated M. tuberculosis H37Ra. Two-dimensional gel electrophoresis was performed to separate culture supernatant proteins extracted from M. tuberculosis H37Rv and M. tuberculosis H37Ra. The protein spots of interest were identified by mass spectrometry, and then the genes encoding the identified proteins were cloned and sequenced. Comparison of silver-stained gels showed that three well-resolved protein spots were present in M. tuberculosis H37Rv but absent from M. tuberculosis H37Ra. Protein spot no. 1 was identified as Rv2346c. Protein spot no. 2 was identified as Rv2347c, Rv1197, Rv1038c, and Rv3620c, which shared significant homology and had the same peptide fingerprinting using tryptic digestion. No M. tuberculosis protein matched protein spot no. 3. Rv2346c, Rv2347c, Rv1038c, and Rv3620c of M. tuberculosis H37Rv were located on the M. tuberculosis H37Ra chromosome, and multiple mutations were observed in the corresponding areas of M. tuberculosis H37Ra. Codon 59 (CAG, Gln) of Rv2347c and Rv3620c was replaced by termination codon (TAG) in M. tuberculosis H37Ra, which probably terminated the polypeptide elongation. These results demonstrate the importance of studying the gene products of M. tuberculosis and show that subtle differences in isogenic mutant strains might play an important role in identifying the attenuating mutations.

Bacterial Proteins↗
PubMed2006

Comparative proteomics of cerebrospinal fluid in neuropathologically-confirmed Alzheimer's disease and non-demented elderly subjects.

OBJECTIVES: Diagnostic tests able to reveal Alzheimer's disease (AD) in living patients before cognitive ability is destroyed are urgently needed. Such tests must distinguish AD from other dementia causes, as well as differentiate subtle changes associated with normal aging from true pathology emergence. A single biomarker offering such diagnostic and prognostic capacities has eluded identification. Therefore, a valuable test for AD is likely to be based on a specific pattern of change in a set of proteins, rather than a single protein. METHODS: We examined pooled cerebrospinal fluid (CSF) samples obtained from neuropathologically-confirmed AD (n=43) and non-demented control subjects (n=43) using 2-dimensional gel electrophoresis (2DE) proteomic methodology to detect differentially expressed proteins. Proteins exhibiting expression level differences between the pools were recovered and identified using matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry. RESULTS: Five differentially-expressed proteins with potential roles in amyloid-beta metabolism and vascular and brain physiology [apolipoprotein A-1 (Apo A-1), cathepsin D (CatD), hemopexin (HPX), transthyretin (TTR), and two pigment epithelium-derived factor (PEDF) isoforms] were identified. Apo A-1, CatD and TTR were significantly reduced in the AD pool sample, while HPX and the PEDF isoforms were increased in AD CSF. DISCUSSION: These results suggest that multi-factor proteomic pattern analysis of the CSF may provide a means to diagnose and assess AD.

Aged↗
PubMed2006

Comparative proteomic analysis of extracellular matrix proteins secreted by two types of skin fibroblasts.

The hair follicle dermal papilla is composed primarily of extracellular matrix (ECM) proteins secreted by resident fibroblasts. Dermal papilla is endowed with hair morphogenic properties, yet its composition is poorly characterized. In an attempt to understand its specificity better, we compared the protein composition of ECM secreted by cultured dermal papilla fibroblasts with that of dermal fibroblasts. ECM proteins are generally large, difficult to solubilize, and abundantly post-translationally modified. We thus implemented an original protocol for analyzing them: ECM samples were enzymatically digested directly in the culture flasks and analyzed by LC-MS/MS. Sequencing of proteolytic peptides by MS/MS yielded protein identification. The relative abundance of a given protein in dermal fibroblast versus dermal papilla samples was estimated by comparing proteolytic peptide intensities detected by MS. Using this approach, several matrix proteins were found to be present at markedly different levels in each ECM type; in particular, thrombospondin 1 and fibronectin appeared to be overrepresented in the dermal papilla fibroblast ECM. MS results were supported by Western blot and immunostaining experiments. In addition, peptide intensities were processed in two ways, which proved to favor either the quantification accuracy or the information precision at the sequence level.

Blotting, Western↗
PubMed2003

Sodium dodecyl sulfate versus acid-labile surfactant gel electrophoresis: comparative proteomic studies on rat retina and mouse brain.

A long-chain derivative of 1,3-dioxolane sodium propyloxy sulfate, with similar denaturing and electrophoretic properties as SDS, and facilitated protein identification following polyacrylamide gel electrophoresis (PAGE) for Coomassie-stained protein bands, has been tested. Comparative acid-labile surfactant/sodium dodecyl sulfate two-dimensional (ALS/SDS 2-D)-PAGE experiments of lower abundant proteins from the proteomes of regenerating rat retina and mouse brain show that peptide recovery for mass spectrometry (MS) mapping is significantly enhanced using ALS leading to more successful database searches. ALS may influence some procedures in proteomic analysis such as the determination of protein content and methods need to be adjusted to that effect. The promising results of the use of ALS in bioanalytics call for detailed physicochemical investigations of surfactant properties.

Acids↗
PubMed2006

Comparative proteomics of symbiotic and aposymbiotic juvenile soft corals.

The symbiotic association between corals and photosynthetic unicellular algae is of great importance in coral reef ecosystems. The study of symbiotic relationships is multidisciplinary and involves research in phylogeny, physiology, biochemistry, and ecology. An intriguing phase in each symbiotic relationship is its initiation, in which the partners interact for the first time. The examination of this phase in coral-algae symbiosis from a molecular point of view is still at an early stage. In the present study we used 2-dimensional polyacrylamide gel electrophoresis to compare patterns of proteins synthesized in symbiotic and aposymbiotic primary polyps of the Red Sea soft coral Heteroxenia fuscescens. This is the first work to search for symbiosis-specific proteins during the natural onset of symbiosis in early host ontogeny. The protein profiles reveal changes in the host soft coral proteome through development, but surprisingly virtually no changes in the host proteome as a function of symbiotic state.

Algal Proteins↗
PubMed2006

Comparative proteomic analysis of Staphylococcus aureus strains with differences in resistance to the cell wall-targeting antibiotic vancomycin.

Three isogenic strains derived from a clinical vancomycin-intermediate Staphylococcus aureus isolate were examined by comparative protein abundance analysis. Subcellular fractionation was followed by protein separation in 2-DE gels and spot identification by MALDI-TOFTOF-MS and LC-MS/MS. Sixty-five significant protein abundance changes were determined. Numerous enzymes participating in the purine biosynthesis pathway were dramatically increased in abundance in strain VP32, which featured the highest minimal inhibitory concentration for vancomycin, compared to strains P100 and HIP5827. Peptidoglycan hydrolase LytM (LytM) and the SceD protein, a putative transglycosylase, were increased in abundance in the cell envelope fraction of strain VP32, whereas the enzyme D-Ala-D-Ala ligase was decreased in its cytosol fraction. Furthermore, penicillin-binding protein 2 (PBP2) had substantially higher activity in strain VP32 compared to that in strain HIP5827. LytM, PBP2 and D-Ala-D-Ala ligase catalyze reactions in the biosynthesis or the metabolism of cell wall peptidoglycan. It is plausible that expression and activity changes of these enzymes in strain VP32 are responsible for an altered cell wall turnover rate, which has been observed, and an altered peptidoglycan structure, which has yet to be elucidated for this highly vancomycin-resistant strain.

Amino Acid Sequence↗
PubMed2006

Comparative proteome analysis of changes in the 26S proteasome during oocyte maturation in goldfish.

Proteasomes are large, multi-subunit particles that act as the proteolytic machinery for most of the regulated intracellular protein degradation in eukaryotic cells. An alteration of proteasome function may be important for the regulation of the meiotic cell cycle. To study the change at the subunit level of the 26S proteasome during meiotic maturation, we purified 26S proteasomes from immature and mature oocytes of goldfish. Two-dimensional polyacrylamide gel electrophoresis was used to separate proteins. For differential analysis, whole spots of the 26S proteasome from goldfish oocytes were identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry and database analysis. Four spots that were different (only detected in mature oocyte 265 proteasomes and not in immature ones) and four protein spots that were up- or down-regulated were identified unambiguously. The mature-specific spots were not 26S proteasome components but rather their interacting proteins, and were identified as chaperonin-containing TCP-1 subunits and myosin light chain. Minor spots of three subunits of the 20S core particle and one of the 19S regulatory particle showed meiotic cell cycle-dependent changes. These results demonstrate that modifications of proteasomal subunits and cell cycle phase-dependent interactions of proteins with proteasomes occur during oocyte maturation in goldfish.

Animals↗
PubMed2006

Comparative proteomics of Dehalococcoides spp. reveals strain-specific peptides associated with activity.

Anaerobic reductive dehalogenation by Dehalococcoides spp. is an ideal system for studying functional diversity of closely related strains of bacteria. In Dehalococcoides spp., reductive dehalogenases (RDases) are key respiratory enzymes involved in the anaerobic detoxification of halogenated compounds at contaminated sites globally. Although housekeeping genes sequenced from Dehalococcoides spp. are >85% identical at the amino acid level, different strains are capable of dehalogenating diverse ranges of compounds, depending largely on the suite of RDase genes that each strain harbors and expresses. We identified RDase proteins that corresponded to known functions in four characterized cultures and predicted functions in an uncharacterized Dehalococcoides-containing mixed culture. Homologues within RDase subclusters containing PceA, TceA, and VcrA were among the most frequently identified proteins. Several additional proteins, including a formate dehydrogenase-like protein (Fdh), had high coverage in all strains and under all growth conditions.

Amino Acid Sequence↗
PubMed2004

Comparative proteomics and subtyping of venom phospholipases A2 and disintegrins of Protobothrops pit vipers.

To explore the venom diversity and systematics of pit vipers under the genus Protobothrops, the venom phospholipases A2 (PLA2s) of P. mangshanensis, P. elegans and P. tokarensis were purified and characterized for the first time. The results were compared with the corresponding venom data of other co-generic species including P. mucrosquamatus, P. flavoviridis and P. jerdonii. Based on sequence features at the N-terminal regions, we identified five PLA2 subtypes, i.e., the Asp49-PLA2s with N6, E6 or R6 substitution and the Lys49-PLA2. However, not all subtypes were expressed in each of the species. Venom N6-PLA2s from P. mangshanensis and P. tokarensis venom were weakly neurotoxic toward chick biventer cervicis tissue preparations. The venoms of P. tokarensis and P. flavoviridis contained identical PLA2 isoforms. In most Protobothrop disintegrins, sequences flanking the RGD-motif are conserved. Phylogenetic analyses based on amino acid sequences of both families of the acidic PLA2s and the disintegrins clarify that these species could belong to a monophyletic group.

Amino Acid Sequence↗
PubMed2003

Comparative proteomics analysis of human lung squamous carcinoma.

Two-dimensional polyacrylamide gel electrophoresis (2-DE) profiles of human lung squamous carcinoma tissue and paired surrounding normal bronchial epithelial tissue were compared. Selected differential protein-spots were identified with peptide mass fingerprinting based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and database searching. Well-resolved and reproducible 2-DE patterns of both the tumor and the normal tissues were acquired. The average deviations of spot position were 0.873+/-0.125mm in IEF direction and 1.025+/-0.213mm in SDS-PAGE direction, respectively. For the tumor tissues, a total of 1349+/-67 spots were detected and 1235+/-48 spots were matched with an average matching rate of 91.5%. For the corresponding normal tissues, a total of 1297+/-73 spots were detected and 1183+/-56 spots were matched with an average matching rate of 91.2%. A total of 1069+/-45 spots were matched between the tumor and the normal tissues. Forty differential proteins between tumor and normal tissues were characterized. Some proteins were the products of oncogenes and others were involved in the regulation of cell cycle and signal transduction. These data are valuable for mass identification of differentially expressed proteins involved in lung carcinogenesis, establishing human lung cancer proteome database and screening molecular marker to further study human lung squamous carcinoma.

Carcinoma, Squamous Cell↗
PubMed2002

Comparative proteome analysis of Chlamydia trachomatis serovar A, D and L2.

Chlamydia trachomatis represents a group of human pathogenic obligate intracellular and gram-negative bacteria. The genome of C. trachomatis D comprises 894 open reading frames (ORFs). In this study the global expression of genes in C. trachomatis A, D and L2, which are responsible for different chlamydial diseases, was investigated using a proteomics approach. Based on silver stained two-dimensional polyacrylamide gel electrophoresis (2-D PAGE), gels with purified elementary bodies (EB) and auto-radiography of gels with 35S-labeled C. trachomatis proteins up to 700 protein spots were detectable within the range of the immobilized pH gradient (IPG) system used. Using mass spectrometry and N-terminal sequencing followed by database searching we identified 250 C. trachomatis proteins from purified EB of which 144 were derived from different genes representing 16% of the ORFs predicted from the C. trachomatis D genome and the 7.5 kb C. trachomatis plasmid. Important findings include identification of proteins from the type III secretion apparatus, enzymes from the central metabolism and confirmation of expression of 25 hypothetical ORFs and five polymorphic membrane proteins. Comparison of serovars generated novel data on genetic variability as indicated by electrophoretic variation and potentially important examples of serovar specific differences in protein abundance. The availability of the complete genome made it feasible to map and to identify proteins of C. trachomatis on a large scale and the integration of our data in a 2-D PAGE database will create a basis for post genomic research, important for the understanding of chlamydial development and pathogenesis.

Autoradiography↗
PubMed2004

Comparative proteome analysis of Mycobacterium tuberculosis grown under aerobic and anaerobic conditions.

Data are presented from two-dimensional (2-D) PAGE analysis of Mycobacterium tuberculosis strain Harlingen grown during aerobic and anaerobic culture, according to a modified Wayne dormancy model. M. tuberculosis cultures were grown to the transition point between exponential growth and stationary phase in the presence of oxygen (7 days) and then part of the cultures was shifted to anaerobic conditions for 16 days. Growth declined similarly during aerobic and anaerobic conditions, whereas the ATP consumption rapidly decreased in the anaerobic cultures. 2-D PAGE revealed 50 protein spots that were either unique to, or more abundant during, anaerobic conditions and 16 of these were identified by MALDI-TOF. These proteins were the alpha-crystalline homologue (HspX), elongation factor Tu (Tuf), GroEL2, succinyl-CoA : 3-oxoacid-CoA transferase (ScoB), mycolic acid synthase (CmaA2), thioredoxin (TrxB2), beta-ketoacyl-ACP synthase (KasB), l-alanine dehydrogenase (Ald), Rv2005c, Rv2629, Rv0560c, Rv2185c and Rv3866. Some protein spots were found to be proteolytic fragments, e.g. HspX and GroEL2. These data suggest that M. tuberculosis induces expression of about 1 % of its genes in response to dormancy.

3-Oxoacyl-(Acyl-Carrier-Protein) Synthase↗
PubMed2006

Comparative proteome approach to characterize the high-pressure stress response of Lactobacillus sanfranciscensis DSM 20451(T).

High hydrostatic pressure (HHP) exerts diverse effects on microorganisms, leading to stress response and cell death. While inactivation of microorganisms by lethal HHP is well investigated in the context of food preservation and the hygienic safety of minimal food processes, sublethal HHP stress response and its effect on adaptation and cross-protection is less understood. In this study, the HHP stress response of Lactobacillus sanfranciscensis was characterized and compared with cold, heat, salt, acid and starvation stress at the proteome level by using 2-DE so as to provide insight into general versus specific stress responses. Sixteen proteins were found to be affected by HHP and were identified by using N-terminal amino acid sequencing and MS. Only one slightly increased protein was specific to the HHP response and showed homology to a clp protease. The other proteins were influenced by most of the investigated stresses in a similar way as HHP. The highest similarity in the HHP proteome was found to be with cold- and NaCl-stressed cells, with 11 overlapping proteins. At the proteome level, L. sanfranciscensis appears to use overlapping subsets of stress-inducible proteins rather than stereotype responses. Our data suggest that a specific pressure response does not exist in this bacteria.

Amino Acid Sequence↗
PubMed2006

Comparative proteomic analysis of high cell density cultivations with two recombinant Bacillus megaterium strains for the production of a heterologous dextransucrase.

High cell density cultivations were performed under identical conditions for two Bacillus megaterium strains (MS941 and WH320), both carrying a heterologous dextransucrase (dsrS) gene under the control of the xylA promoter. At characteristic points of the cultivations (end of batch, initial feeding, before and after induction) the proteome was analyzed based on two dimensional gel electrophoresis and mass spectrometric protein identification using the protein database "bmegMEC.v2" recently made available. High expression but no secretion of DsrS was found for the chemical mutant WH320 whereas for MS 941, a defined protease deficient mutant of the same parent strain (DSM319), not even expression of DsrS could be detected. The proteomic analysis resulted in the identification of proteins involved in different cellular pathways such as in central carbon and overflow metabolism, in protein synthesis, protein secretion and degradation, in cell wall metabolism, in cell division and sporulation, in membrane transport and in stress responses. The two strains exhibited considerable variations in expression levels of specific proteins during the different phases of the cultivation process, whereas induction of DsrS production had, in general, little effect. The largely differing behaviour of the two strains with regard to DsrS expression can be attributed, at least in part, to changes observed in the proteome which predominantly concern biosynthetic enzymes and proteins belonging to the membrane translocation system, which were strongly down-regulated at high cell densities in MS941 compared with WH320. At the same time a cell envelope-associated quality control protease and two peptidoglycan-binding proteins related to cell wall turnover were strongly expressed in MS941 but not found in WH320. However, to further explain the very different physiological responses of the two strains to the same cultivation conditions, it is necessary to identify the mutated genes in WH320 in addition to the known lacZ. In view of the results of this proteomic study it seems that at high cell density conditions and hence low growth rates MS941, in contrast to WH320, does not maintain a vegetative growth which is essential for the expression of the foreign dsrS gene by using the xylA promoter. It is conceivable that applications of a promoter which is highly active under nutrient-limited cultivation conditions is necessary, at least for MS941, for the overexpression of recombinant genes in such B. megaterium fed-batch cultivation process. However to obtain a heterologous protein in secreted and properly folded form stills remains a big challenge.

Journal Article↗