Extremely diversified haplotypes observed among assemblage B population of Giardia intestinalis in Kenya.

In molecular epidemiological studies of Giardia intestinalis, an pathogenic intestinal flagellate, due to the presence of allelic sequence heterogeneity (ASH) on the tetraploid genome, the image of haplotype diversity in the field remains uncertain. Here we employed the nine assemblage B positive stool samples, which had previously reported from Kenyan children, for the clonal sequence analysis of multiple gene loci (glutamate dehydrogenase (GDH), triosephosphate isomerase (TPI), and beta-giardin (BG)). The diversified unique assemblage B haplotypes as GDH (n = 67), TPI (n = 84), and BG (n = 62), and the assemblage A haplotypes as GDH (n = 7), TPI (n = 14), and BG (n = 15), which were hidden in the previous direct-sequence results, were detected. Among the assemblage B haplotypes, Bayesian phylogeny revealed multiple statistically significant clusters (9, 7, and 7 clusters for GDH, TPI, and BG, respectively). A part of the clusters (2 for GDH and 1 for BG), which included >4 haplotypes from an individual sample, indicated the presence of co-transmission with multiple strains sharing a recent ancestor. Locus-dependent discrepancies, such as different compositions of derived samples in clusters and different genotyping results for the assemblages, were also observed and considered to be the traces of both intra- and inter-assemblage genetic recombination respectively. Our clonal sequence analysis for giardial population, which applied firstly in Kenya, could reveal the higher rates of ASH far beyond the levels reported in other areas and address the complex population structure. The clonal analysis is indispensable for the molecular field study of G. intestinalis.

Initial detections and spread of invasive Spodoptera frugiperda in China and comparisons with other noctuid larvae in cornfields using molecular techniques.

The fall armyworm, Spodoptera frugiperda, is a species native to the Americas and has spread to many countries in Africa and Asia in recent years. Proactive actions for potential invasion of S. frugiperda to China coordinated by government agencies and agricultural extension systems resulted in timely detection in January 2019 in Yunnan province neighboring onto Myanmar. The extensive monitoring in southern provinces of China since February 2019 resulted in dynamic tracking of S. frugiperda spreading to 13 provincial regions in China within 4 months by May 10, 2019, which is crucial for timely management actions in the fields. The first detections of S. frugiperda (corn strain) in China were confirmed using cytochrome oxidase subunit 1 (CO1) and triosephosphate isomerase (Tpi) genes molecular marker method. In addition to S. frugiperda, larvae of three other noctuid species with similar morphological appearance (S. litura, S. exigua and Mythimna separata) can occur simultaneously and cause similar damage in cornfields in southern China. Thus, we can use both morphological and molecular marker methods to compare larval stages of four noctuid species. Further, we discuss the risk of potential spread of invasive S. frugiperda to other regions and impact on corn production in China.

Differential Protein Expressions in Virus-Infected and Uninfected Trichomonas vaginalis.

Protozoan viruses may influence the function and pathogenicity of the protozoa. Trichomonas vaginalis is a parasitic protozoan that could contain a double stranded RNA (dsRNA) virus, T. vaginalis virus (TVV). However, there are few reports on the properties of the virus. To further determine variations in protein expression of T. vaginalis, we detected 2 strains of T. vaginalis; the virus-infected (V+) and uninfected (V-) isolates to examine differentially expressed proteins upon TVV infection. Using a stable isotope N-terminal labeling strategy (iTRAQ) on soluble fractions to analyze proteomes, we identified 293 proteins, of which 50 were altered in V+ compared with V- isolates. The results showed that the expression of 29 proteins was increased, and 21 proteins decreased in V+ isolates. These differentially expressed proteins can be classified into 4 categories: ribosomal proteins, metabolic enzymes, heat shock proteins, and putative uncharacterized proteins. Quantitative PCR was used to detect 4 metabolic processes proteins: glycogen phosphorylase, malate dehydrogenase, triosephosphate isomerase, and glucose-6-phosphate isomerase, which were differentially expressed in V+ and V- isolates. Our findings suggest that mRNA levels of these genes were consistent with protein expression levels. This study was the first which analyzed protein expression variations upon TVV infection. These observations will provide a basis for future studies concerning the possible roles of these proteins in host-parasite interactions.

Selective Reaction Monitoring Mass Spectrometry for Quantitation of Glycolytic Enzymes in Postmortem Brain Samples.

Patients with psychiatric disorders exhibit dysfunctions in peripheral and central metabolism. This may be a root cause of impaired neuronal function, manifested as changes in mood, behavior, and cognitive capabilities in patients suffering with these conditions. Here we describe a selective reaction monitoring mass spectrometry (SRM-MS)-based targeted proteomic protocol for precise simultaneous quantitation of three glycolytic enzymes in postmortem brain tissue extracts. The SRM-MS approach has several advantages in terms of sensitivity, reproducibility, and reduced sample consumption, compared to traditional MS methods.

Protein markers for discrimination of meat species in raw beef, pork and poultry and their mixtures.

The purpose of this study was to find discrimination markers for four major meat species such as beef, pork, chicken and duck. Myofibrillar and sarcoplasmic proteins isolated from each meat type were analyzed by one-dimensional gel electrophoresis and some proteins were identified through LC-MS/MS analysis. We confirmed that troponin I (TnI), enolase 3, l-lactate dehydrogenase (LDH) and triose-phosphate isomerase (TPI) could be useful markers for discrimination of mammals from poultry due to their different electrophoretic mobility. Tropomyosin 1 and carbonic anhydrase 3 were observed as muscle fiber type-related proteins and these could also be markers to distinguish mammals from poultry. Species-specific peptides identified by LC-MS/MS spectra allow the identification of each species regardless of the same protein. Therefore, it is easy to discriminate between mammals and poultry by comparing the electrophoretic mobility of TnI, enolase 3, LDH, TPI and CA3, and each species could be identified through LC-MS/MS analysis.

Differential proteomic analysis of embryogenic lines in oil palm (Elaeis guineensis Jacq).

UNLABELLED: Oil palm tissue culture is one way to produce superior oil palm planting materials. However, the low rate of embryogenesis is a major hindrance for the adoption of this technology in oil palm tissue culture laboratories. In this study, we use proteomic technologies to compare differential protein profiles in leaves from palms of high and low proliferation rates in tissue culture in order to understand the underlying biological mechanism for the low level of embryogenesis. Two protein extraction methods, namely trichloroacetic acid/acetone precipitation and polyethylene glycol fractionation were used to produce total proteins and fractionated protein extracts respectively, with the aim of improving the resolution of protein species using two-dimensional gel electrophoresis. A total of 40 distinct differential abundant protein spots were selected from leaf samples collected from palms with proven high and low proliferation rates. The variant proteins were subsequently identified using mass spectrometric analysis. Twelve prominent protein spots were then characterised using real-time polymerase chain reaction to compare the mRNA expression and protein abundant profiles. Three proteins, namely triosephosphate isomerase, l-ascorbate peroxidase, and superoxide dismutase were identified to be potential biomarker candidates at both the protein abundant and mRNA expression levels. BIOLOGICAL SIGNIFICANCE: In this study, proteomic analysis was used to identify abundant proteins from total protein extracts. PEG fractionation was used to reveal lower abundant proteins from both high and low proliferation embryogenic lines of oil palm samples in tissue culture. A total of 40 protein spots were found to be significant in abundance and the mRNA levels of 12 of these were assessed using real time PCR. Three proteins namely, triosephosphate isomerase, l-ascorbate peroxidase and superoxide dismutase were found to be concordant in their mRNA expression and protein abundance. Triosephosphate isomerase is a key enzyme in glycolysis. Both l-ascorbate peroxidase and superoxide dismutase play a role in anti-oxidative scavenging defense systems. These proteins have potential for use as biomarkers to screen for high and low embryogenic oil palm samples.

Development of a non-denaturing 2D gel electrophoresis protocol for screening in vivo uranium-protein targets in Procambarus clarkii with laser ablation ICP MS followed by protein identification by HPLC-Orbitrap MS.

Limited knowledge about in vivo non-covalent uranium (U)-protein complexes is largely due to the lack of appropriate analytical methodology. Here, a method for screening and identifying the molecular targets of U was developed. The approach was based on non-denaturing 1D and 2D gel electrophoresis (ND-PAGE and ND-2D-PAGE (using ND-IEF as first dimension previously described)) in conjunction with laser ablation inductively coupled plasma mass spectrometry (LA-ICP MS) for the detection of U-containing proteins. The proteins were then identified by µbore HPLC-Orbitrap MS/MS. The method was applied to the analysis of cytosol of hepatopancreas (HP) of a model U-bioaccumulating organism (Procambarus clarkii). The imaging of uranium in 2D gels revealed the presence of 11 U-containing protein spots. Six protein candidates (i.e. ferritin, glyceraldehyde-3-phosphate dehydrogenase, triosephosphate isomerase, cytosolic manganese superoxide dismutase (Mn-SOD), glutathione S transferase D1 and H3 histone family protein) were then identified by matching with the data base of crustacea Decapoda species (e.g. crayfish). Among them, ferritin was the most important one. This strategy is expected to provide an insight into U toxicology and metabolism.

Characterization and proteome analysis of inosine 5-monophosphate dehydrogenase in epidemic Streptococcus suis serotype 2.

Streptococcus suis serotype 2 (SS2) is an important zoonotic pathogen that causes severe disease symptoms in pigs and humans. In the present study, we found one isogenic mutant lacking inosine 5-monophosphate dehydrogenase (IMPDH) ΔZY05719 was attenuated in pigs compared with the wild-type SS2 strain ZY05719. Comparative proteome analysis of the secreted proteins expression profiles between ZY05719 and ΔZY05719 allowed us to identify Triosephosphate isomerase (TPI) and glyceraldehyde phosphate dehydrogenase (GAPDH), which were down expressed in the absence of the IMPDH. Both of them are glycolytic enzymes participating in the glycolytic pathway. Compared with ZY05719, ΔZY05719 lost the ability of utilize mannose, which might relate to down expression of TPI and GAPDH. In addition, GAPDH is a well-known factor that involved in adhesion to host cells, and we demonstrated ability of adhesion to HEp-2 and PK15 by ΔZY05719 was significantly weakened, in contrast to ZY05719. The adhesion to host cells is the crucial step to cause infection for pathogen, and the reduction adhesion of ΔZY05719, to some extent illustrates the attenuated virulence of ΔZY05719.

Localization by scanning immunoelectron microscopy of triosephosphate isomerase, the molecules responsible for contact-mediated killing of Cryptococcus, on the surface of Staphylococcus.

T In our previous studies, TPI were found to be the molecules responsible for contact-killing of C. neoformans by S. aureus cells. Since TPI is a glycolytic protein that functions in the cytoplasm, evidence that TPI is present on the surface of S. aureus was required. In the present study, the presence of TPI on the cell surface of S. aureus was demonstrated by agglutination test and scanning immunoelectron microscopy. Furthermore, TPI was found to be present at a lower density than protein A/G molecules on the surface of S. aureus.

TIM-Finder: a new method for identifying TIM-barrel proteins.

BACKGROUND: The triosephosphate isomerase (TIM)-barrel fold occurs frequently in the proteomes of different organisms, and the known TIM-barrel proteins have been found to play diverse functional roles. To accelerate the exploration of the sequence-structure protein landscape in the TIM-barrel fold, a computational tool that allows sensitive detection of TIM-barrel proteins is required. RESULTS: To develop a new TIM-barrel protein identification method in this work, we consider three descriptors: a sequence-alignment-based descriptor using PSI-BLAST e-values and bit scores, a descriptor based on secondary structure element alignment (SSEA), and a descriptor based on the occurrence of PROSITE functional motifs. With the assistance of Support Vector Machine (SVM), the three descriptors were combined to obtain a new method with improved performance, which we call TIM-Finder. When tested on the whole proteome of Bacillus subtilis, TIM-Finder is able to detect 194 TIM-barrel proteins at a 99% confidence level, outperforming the PSI-BLAST search as well as one existing fold recognition method. CONCLUSIONS: TIM-Finder can serve as a competitive tool for proteome-wide TIM-barrel protein identification. The TIM-Finder web server is freely accessible at http://202.112.170.199/TIM-Finder/.

Identification of proteins with increased expression in rheumatoid arthritis synovial tissues.

OBJECTIVE: A proteomic approach was applied to discover novel rheumatoid arthritis (RA)-specific proteins by comparing the expression profiles of synovial membranes from patients with RA, osteoarthritis (OA), and ankylosing spondylitis (AS). METHODS: Synovial tissues were collected from patients with RA (n = 10), OA (n = 10), or AS (n = 6), and healthy controls matched for age and sex. Proteins were separated by 2-dimensional polyacrylamide gel electrophoresis, and the proteins with significantly increased expression in the RA samples were subject to matrix-assisted laser adsorption-ionization time-of-flight spectrometry. Results were verified using Western blot and immunohistochemistry. Levels of the candidate proteins were measured within plasma and synovial fluids from the RA patients (n = 30), who had disease duration of 3-7 years, using ELISA. Levels were also measured within plasma from unmedicated RA patients (n = 41), who had disease duration of 1-6 months. RESULTS: Compared with the OA and AS tissue samples, the proteins Ig-kappa light-chain C region, PRDX4, SOD2, TPI, and TXNDC5 were found with increased expression in synovial tissues of RA patients. PRDX4, SOD2, TPI, and TXNDC5 had 2-fold or more increase in expression in some of the early RA plasma samples (58.55%, 31.7%, 26.8%, and 36.6%, respectively) as compared with the early OA samples and control samples. TXNDC5 had 2-fold or more increase in expression in 53.3% of blood samples and 73.3% of synovial fluid samples from patients with long disease duration of RA as compared with samples from OA and AS patients. CONCLUSION: Functional classification indicated that these identified proteins were related with cell differentiation, glycol metabolism, immunoactivation, and endogenous antioxidant reaction.

Amyloid-dependent triosephosphate isomerase nitrotyrosination induces glycation and tau fibrillation.

Alzheimer's disease neuropathology is characterized by neuronal death, amyloid beta-peptide deposits and neurofibrillary tangles composed of paired helical filaments of tau protein. Although crucial for our understanding of the pathogenesis of Alzheimer's disease, the molecular mechanisms linking amyloid beta-peptide and paired helical filaments remain unknown. Here, we show that amyloid beta-peptide-induced nitro-oxidative damage promotes the nitrotyrosination of the glycolytic enzyme triosephosphate isomerase in human neuroblastoma cells. Consequently, nitro-triosephosphate isomerase was found to be present in brain slides from double transgenic mice overexpressing human amyloid precursor protein and presenilin 1, and in Alzheimer's disease patients. Higher levels of nitro-triosephosphate isomerase (P < 0.05) were detected, by Western blot, in immunoprecipitates from hippocampus (9 individuals) and frontal cortex (13 individuals) of Alzheimer's disease patients, compared with healthy subjects (4 and 9 individuals, respectively). Triosephosphate isomerase nitrotyrosination decreases the glycolytic flow. Moreover, during its isomerase activity, it triggers the production of the highly neurotoxic methylglyoxal (n = 4; P < 0.05). The bioinformatics simulation of the nitration of tyrosines 164 and 208, close to the catalytic centre, fits with a reduced isomerase activity. Human embryonic kidney (HEK) cells overexpressing double mutant triosephosphate isomerase (Tyr164 and 208 by Phe164 and 208) showed high methylglyoxal production. This finding correlates with the widespread glycation immunostaining in Alzheimer's disease cortex and hippocampus from double transgenic mice overexpressing amyloid precursor protein and presenilin 1. Furthermore, nitro-triosephosphate isomerase formed large beta-sheet aggregates in vitro and in vivo, as demonstrated by turbidometric analysis and electron microscopy. Transmission electron microscopy (TEM) and atomic force microscopy studies have demonstrated that nitro-triosephosphate isomerase binds tau monomers and induces tau aggregation to form paired helical filaments, the characteristic intracellular hallmark of Alzheimer's disease brains. Our results link oxidative stress, the main etiopathogenic mechanism in sporadic Alzheimer's disease, via the production of peroxynitrite and nitrotyrosination of triosephosphate isomerase, to amyloid beta-peptide-induced toxicity and tau pathology.

Biochemical and functional characterization of triosephosphate isomerase from Mycobacterium tuberculosis H37Rv.

Triosephosphate isomerase (TPI), one of the key enzymes of the glycolytic pathway, is an attractive drug target against Mycobacterium tuberculosis as glycolysis provides the majority of the organism's energy requirements inside macrophages. To carry out biochemical and biophysical characterization, purified recombinant M. tuberculosis TPI produced in Escherichia coli was used. Mass spectrum analysis showed M. tuberculosis rTPI to be of 28 213 Da. The biologically active enzyme is a homodimer as determined by gel filtration chromatography. The M. tuberculosis TPI had a pH optimum in the range of 6-8 and a temperature optimum around 37 degrees C. Circular dichroism spectra analysis revealed that loss of secondary structure of rTPI occurs around 60 degrees C. Metal cations were not required for M. tuberculosis TPI activity. The k(cat) was 4.1 x 10(6) min(-1). Importantly, the apparent K(m) value of M. tuberculosis rTPI for the substrate glyceraldehyde-3-phosphate is 84 microM which is sevenfold higher than the value reported for human TPI. The difference in K(m) is indicative of the difference in the active site of the human and M. tuberculosis TPI, which can be exploited for drug designing specifically targeting M. tuberculosis TPI.

Retinal proteins modified by 4-hydroxynonenal: identification of molecular targets.

The reactive aldehyde, 4-hydroxynonenal (HNE), is a product of lipid peroxidation that can covalently modify and inactivate proteins. Previously, we reported increased HNE modification of select retinal proteins resolved by one-dimensional gel electrophoresis in aged Fisher 344 x Brown Norway rats (Louie, J.L., Kapphahn, R.J., Ferrington, D.A., 2002. Proteasome function and protein oxidation in the aged retina. Exp. Eye Res. 75, 271-284). In the current study, quantitative assessment of HNE molar content using slot blot immunoassays showed HNE content is increased 30% in aged rat retina. In contrast, there was no age-related difference in HNE content in individual spots resolved by 2D gel electrophoresis suggesting the increased modification is likely on membrane proteins that are missing on 2D gels. The HNE-immunoreactive proteins resolved by 2D gel electrophoresis were identified by MALDI-TOF mass spectrometry. These proteins are involved in metabolism, chaperone function, and fatty acid transport. Proteins that were frequently modified and had the highest molar content of HNE included triosephosphate isomerase, alpha enolase, heat shock cognate 70 and betaB2 crystallin. Immunochemical detection of HNE adducts on retinal sections showed greater immune reaction in ganglion cells, photoreceptor inner segment, and the inner plexiform layer. Identification of HNE modified proteins in two alternative model systems, human retinal pigment epithelial cells in culture (ARPE19) and human donor eyes, indicated that triosephosphate isomerase and alpha enolase are generally modified. These results identify a common subset of proteins that contain HNE adducts and suggest that select retinal proteins are molecular targets for HNE modification.

An unusual triosephosphate isomerase from the early divergent eukaryote Giardia lamblia.

Recombinant triosephosphate isomerase from the parasite Giardia lamblia (GlTIM) was characterized and immunolocalized. The enzyme is distributed uniformly throughout the cytoplasm. Size exclusion chromatography of the purified enzyme showed two peaks with molecular weights of 108 and 55 kDa. Under reducing conditions, only the 55-kDa protein was detected. In denaturing gel electrophoresis without dithiothreitol, the enzyme showed two bands with molecular weights of 28 and 50 kDa; with dithiotretitol, only the 28-kDa protein was observed. These data indicate that GlTIM may exist as a tetramer or a dimer and that, in the former, the two dimers are covalently linked by disulfide bonds. The kinetics of the dimer were similar to those of other TIMs. The tetramer exhibited half of the kcat of the dimer without changes in the Km. Studies on the thermal stability and the apparent association constants between monomers showed that the tetramer was slightly more stable than the dimer. This finding suggests the oligomerization is not related to enzyme thermostability as in Thermotoga maritima. Instead, it could be that oligomerization is related to the regulation of catalytic activity in different states of the life cycle of this mesophilic parasite.

Genotypic differentiation of twelve Clostridium species by polymorphism analysis of the triosephosphate isomerase (tpi) gene.

Housekeeping genes encoding metabolic enzymes may provide alternative markers to 16S ribosomal DNA (rDNA) for genotypic and phylogenetic characterization of bacterial species. We have developed a PCR-restriction fragment length polymorphism (PCR-RFLP) assay, targeting the triosephosphate isomerase (tpi) gene, which allows the differentiation of twelve pathogenic Clostridium species. Degenerate primers constructed from alignments of tpi sequences of various gram-positive bacteria allowed the amplification of a 501 bp target region in the twelve Clostridium type strains. A phylogenetic tree constructed from the nucleotidic sequences of these tpi amplicons was well correlated with that inferred from analysis of 16S rDNA gene sequences. The analysis of tpi sequences revealed restriction sites of enzyme AluI that could be species-specific. Indeed, AluI digestion of amplicons from the twelve type strains provided distinct restriction patterns. A total of 127 strains (three to sixteen strains for each species) was further analyzed by PCR-RFLP of the tpi gene, and confirmed that each species could be characterized by one to three restriction types (RTs). The differences between RTs within species could be explained by point mutations in AluI restriction sites of the tpi sequences. PCR-restriction analysis of the tpi gene offers an accurate tool for species identification within the genus Clostridium, and provides an alternative marker to 16S rDNA for phylogenetic analyses.

Plasticity of protein expression during culture of fetal skin cells.

In order to gain insight into the biology of fetal skin during culture, cellular proteins were studied during four culture passages (P00, P01, P04 as well as P10) using high-resolution two-dimensional (2-D) gel electrophoresis and mass spectrometry (MS). Bioinformatic analyses were focused on a region of each gel corresponding to pI between 4 and 8 and M(r) from 8000 to 35 000. In this area, 373 +/- 42 spots were detected (N = 18). Twenty-six spots presented an integrated intensity that increased in the higher passages, whereas five spots showed a progressively lower intensity in subsequent passaging. MS analysis was performed on spots that were unambiguously identified on preparative 2-D gels. Among the 26 spots showing an increased size between P00 and P10, 9 were identified, and corresponded to 3 proteins: (i) peptidyl-prolyl cis-trans isomerase A (P05092; cyclophilin A or cyclosporin A-binding protein), (ii) triosephosphate isomerase (P00938), and (iii) enoyl-CoA hydratase (P30084). Among these nine identified spots, three were absent at P00, but were present at P10. They corresponded to isoforms of peptidyl-prolyl cis-trans isomerase and triosephosphate isomerase, respectively. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses of the acidic isoforms of triosephosphate isomerase showed modifications of cysteine residues to cysteic acid. All these isoforms were clearly present in the skin cells of a 4-year-old child, as well as in skin cells from a 80-year-old man, at P00. These observations probably reflect either an oxidative stress related to cell culture, or, alternatively, maturation, differentiation and the aging of the cells.

Identification of metabolic enzymes in renal cell carcinoma utilizing PROTEOMEX analyses.

PROTEOMEX, an approach which combines conventional proteome analysis with serological screening, is a powerful tool to separate proteins and identify immunogenic components in malignant diseases. By applying this approach, we characterized nine metabolic enzymes which were differentially expressed in renal cell carcinoma (RCC) cell lines and compared their expression profiles to that of normal kidney epithelium cells. Four of these proteins, superoxide dismutase (SODC), triosephosphatase isomerase (TPIS), thioredoxin (THIO) and ubiquitin carboxyl-terminal hydrolase (UBL1) were further analysed for both their constitutive and interferon (IFN)-gamma inducible protein expression pattern in cell lines or tissue specimens derived from RCC or normal kidney epithelium using Western blot analysis and immunohistochemistry, respectively. With the exception of the RCC cell line MZ1940RC, which completely lacks the expression of UBL1, a heterogeneous and variable expression pattern of the different metabolic enzymes was detected in RCC and normal renal epithelium. The highest differences in the expression levels were found for THIO in the RCC cell lines, which was 2-fold upregulated when compared to autologous normal kidney epithelium. Moreover, IFN-gamma treatment did not influence the constitutive expression of these metabolic enzymes. Thus, PROTEOMEX represents a valuable approach for the identification of metabolic enzymes which might be used as markers for the diagnosis of RCC.

Restricted access chromatographic sample preparation of low mass proteins expressed in human fibroblast cells for proteomics analysis.

Two-dimensional electrophoresis and modern image analysis systems have made it possible to study protein expression and regulation of proteins in biological systems. Proteins in the molecular mass region of 20-120 kDa are well investigated and described. However, proteins with masses below 20 kDa are the least investigated as they are rarely seen on 2D-PAGE due to fast migrations in the electric field and lack of staining efficiency. This paper describes a technique that enriches proteins in the lower mass region using solid-phase extraction. The purification step is carried out using C18 functionalised "restricted access" affinity chromatography whereby simultaneous trace enrichment and sample clean up is achieved. In this study expression patterns of TGF-beta stimulated and non-stimulated fibroblasts were compared after the solid-phase fractionation procedure. An increased expression pattern was obtained whereby 400 protein spots could be detected by image analysis in the <20-kDa region. Out of these, specific regulations of 14 spots were found by quantitative image analysis and spots of interest were identified with MALDI TOF-MS. The regulated and identified proteins were triosephosphate isomerase, cofilin and heat shock 27-kDa protein.

"Early" protein synthesis of Lactobacillus delbrueckii ssp. bulgaricus in milk revealed by [35S] methionine labeling and two-dimensional gel electrophoresis.

The proteomes of exponentially growing and stationary cells of Lactobacillus delbrueckii ssp. bulgaricus grown in rich medium (MRS) were separated by two-dimensional polyacrylamide gel electrophoresis (2-DE) and quantified after Coomassie staining. Stationary cells grown in MRS were inoculated in reconstituted skim milk, and "early" protein synthesis during the first 30 min of fermentation in milk was monitored by [35S]methionine labeling and 2-DE. In contrast to exponentially growing or stationary cells, the predominant "early" proteins were small (< 15 kDa) and of low pI (< 5.3). Quantification of the proteome of the "early" lag phase based on 47 "spots" revealed that only three "early" proteins accounted for more than 80% of the total label. They were identified as pI 4.7 and 4.9 isoforms of the heat-stable phosphoryl carrier protein (HPr) with 45.2 and 9.4% of total label, respectively, and an unknown protein called EPr1 ("early" protein 1) with 26.6% of total label. Although an N-terminal sequence of 19 amino acids was obtained, no homologs to EPr1 could be found. De novo synthesis of the 10 and 60 kDa heat shock proteins (GroES and GroEL) was considerably lower (0.04 and 0.9% of total label, respectively), indicating only low levels of stress. Synthesis of triosephosphate isomerase (Tpi) as marker for glycolytic enzymes reached only 0.08% of total label. Our results demonstrate that inoculation in milk, resulting in a change from glucose to lactose as carbon source, imposes only little need for synthesis of stress or glycolytic enzymes, as sufficient proteins are present in the stationary, MRS-grown cells. The high level of expression of the pI 4.7 isoform of HPr suggests a regulatory function of the presumed Ser-46 phosphorylated form of HPr.