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PubMed2006

Closely related dermatophyte species produce different patterns of secreted proteins.

Dermatophytes are the most common infectious agents responsible for superficial mycosis in humans and animals. Various species in this group of fungi show overlapping characteristics. We investigated the possibility that closely related dermatophyte species with different behaviours secrete distinct proteins when grown in the same culture medium. Protein patterns from culture filtrates of several strains of the same species were very similar. In contrast, secreted protein profiles from various species were different, and so a specific signature could be associated with each of the six analysed species. In particular, protein patterns were useful to distinguish Trichophyton tonsurans from Trichophyton equinum, which cannot be differentiated by ribosomal DNA sequencing. The secreted proteases Sub2, Sub6 and Sub7 of the subtilisin family, as well as Mep3 and Mep4 of the fungalisin family were identified. SUB6, SUB7, MEP3 and MEP4 genes were cloned and sequenced. Although the protein sequence of each protease was highly conserved across species, their level of secretion by the various species was not equivalent. These results suggest that a switch of habitat could be related to a differential expression of genes encoding homologous secreted proteins.

Amino Acid Sequence↗

PubMed2004

Proteomic analysis of cleavage events reveals a dynamic two-step mechanism for proteolysis of a key parasite adhesive complex.

The transmembrane micronemal protein MIC2 and its partner M2AP comprise an adhesive complex that is required for rapid invasion of host cells by the obligate intracellular parasite Toxoplasma gondii. Recent studies have shown that the MIC2/M2AP complex undergoes extensive proteolytic processing on the parasite surface during invasion, including primary processing of M2AP by unknown proteases and proteolytic shedding of the complex by an anonymous protease called MPP1. While it was shown that MPP1-mediated cleavage is necessary for efficient invasion, it remained unclear whether the adhesive complex was liberated by juxtamembrane or intramembrane proteolysis. Here, using a three-phase strategy of assigning cleavage sites based on intact matrix-assisted laser desorption/ionization mass followed by confirmation by enzymatic digestion and inhibitor profiling, we demonstrate that M2AP is processed by two parasite-derived proteases called MPP2 and MPP3. We also define the substrate repertoire of MPP2 by two-dimensional differential gel electrophoresis using fluorescent tags. Finally, we use complementary mass spectrometric techniques to unequivocally show that MIC2 is shed by intramembrane cleavage within its anchoring domain. Based on the properties of this cleavage site, we conclude that the sheddase, MPP1, is likely a multipass membrane protease of the Rhomboid family. Our data support a novel two-step proteolysis model that includes primary processing of the MIC2/M2AP complex followed by secondary cleavage to shed the complex from the parasite surface during the final steps of invasion.

Amino Acid Sequence↗

PubMed2026

Integrated multi-omics analysis reveals a pH-driven metabolic and translational switch in Ureaplasma parvum.

Human ureaplasmas are minimal-genome bacteria and pathobionts of the urogenital tract. They must adapt to fluctuating pH conditions despite the absence of canonical transcriptional regulatory systems. However, the mechanisms underlying these responses remain unclear. This study aimed to construct a system-level model of pH adaptation in this minimal pathogen. We used an integrated multi-omics platform combining proteomics, metabolomics, and RNA modification profiling to construct a system-level model of pH adaptation. The results revealed a bifurcated strategy governed by the differential activation of preexisting, co-regulated functional modules. Under neutral pH conditions (pH 7), Ureaplasma parvum activated energy metabolism and upregulated ATP synthesis while forming a stress-counteracting proteostasis pathway. This may suggest a biological energy state under high stress conditions. Conversely, under acidic stress (pH 5), it activated biosynthesis/translation, showing significant upregulation of ribosomal proteins and accumulation of translation precursors and the polyamine spermidine. This may represent a state of expanded translational capacity. This adaptive switch is accompanied by dynamic reorganization of the epitranscriptome, highlighting the importance of post-transcriptional regulation. This study suggests mechanisms by which minimal organisms achieve adaptive plasticity through sophisticated post-transcriptional and metabolic control, providing a new framework for understanding Ureaplasma physiology and the biology of genome-reduced organisms.IMPORTANCEMinimal bacteria challenge canonical views of cellular regulation. In organisms with radically reduced genomes and sparse transcription factors, how adaptive plasticity is achieved remains a core question. Our study proposes a model in which a simple physicochemical cue-extracellular pH-selects among prewired cellular programs, while post-transcriptional and epitranscriptomic layers fine-tune execution. The findings of this study suggest a multi-omics scheme for how organisms adapt to environmental changes and ensure survival without inducing new circuits or complex transcriptional regulation. Conceptually, it proposes regulation via RNA modifications in processes, such as metabolism, proteostasis, and translation. This framework may be generalizable to other genome-reduced microorganisms. Beyond microbiology, it provides design principles for synthetic biology and offers a mechanistic interpretation of phenotypic tolerance to stress factors. It may encourage the use of pH-linked epitranscriptome signals as measurable indicators of cellular state.

Hydrogen-Ion Concentration↗

PubMed2004

[Two-dimensional electrophoregram for proteomic analysis of rat brain with intrahippocampal amyloid beta injection and normal rat brain].

OBJECTIVE: To investigate the molecular mechanisms of Alzheimer's disease by comparing global protein patterns in two-dimensional electrophoregram (2-DE) of the brain of rats with intrahippocampal amyloid beta injection and normal rats. METHODS: From adult SD rats with intrahippocampal injection of amyloid beta, 200 microg brain proteins were extracted with 9 mol/L urea, 4% CHAPS, 1% DTT, 0.5% CA and a cocktail of protease inhibitors. Immobilized pH gradient (IPG) isoelectric focusing electrophoresis of the extracted proteins was performed to obtain the electrophoretogram of the first dimension, with the second dimension obtained by vertical SDS-PAGE. The electrophoretograms were visualized using silver staining and analyzed with ImageMaster 2D-Elite software. RESULTS: On average, 496 and 491 protein spots could be obtained in the electrophoregraphs for rats with amyloid beta and the control rats, respectively, and 30 of these spots exhibited quantitative changes. Another 11 and 6 spots were exclusively shown on the protein maps for amyloid beta-treated rats and control rats, respectively. CONCLUSION: The differentially displayed proteins in the brain identified between the rats with intrahippocampal amyloid beta injection and control rats may provide further insight into the pathogenesis of Alzheimer's disease and useful clues for developing new drugs for its treatment.

Alzheimer Disease↗

PubMed2004

Proteome analysis of intraclonal diversity of two Pseudomonas aeruginosa TB clone isolates.

Two strains, Pseudomonas aeruginosa TB10839 and TB121838, which belong to the TB clonal lineage, have been isolated from sputa of cystic fibrosis patients. Despite the fact that the strains are closely related, their pathogenic potential differs dramatically: while strain TB10839 is capable of proliferating in polymorphonuclear granulocytes, strain TB121838 is not. Comparative two-dimensional polyacrylamide gel electrophoresis coupled to matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF)-mass spectrometry was employed to map the extracellular, intracellular, and surface sub-proteomes of TB10839 and TB121838 and to identify differentially expressed proteins. About 4% of all detected protein spots were differentially expressed between both strains including absent or present spots and spots with a more than 2-fold changed intensity. This percentage reflects a relatively high degree of intraclonal variability. Many of the protein spots in TB10839 that were missing or expressed at lower levels in TB121838 were identified as quorum-sensing regulated virulence factors. It might be speculated that the increased expression of these proteins contributes to pathogenic competence of TB10839.

Clone Cells↗

PubMed2007

Oxidative stress in the aging murine olfactory bulb: redox proteomics and cellular localization.

A recent proteomics analysis from our laboratory demonstrated that several oxidative stress response proteins showed significant changes in steady-state levels in olfactory bulbs (OBs) of 20- vs. 1.5-month-old mice. Oxidative stress may result in protein oxidation. In this study, we investigated two forms of protein oxidative modification in murine OBs: carbonylation and nitration. Redox proteomics with two-dimensional gel electrophoresis, Western blotting, protein digestion, and mass spectrometry was used to quantify total and specific protein carbonylation and to identify differentially carbonylated proteins and determine the carbonylation status of previously identified proteins in OBs of 1.5- and 20-month-old mice. Immunohistochemistry was used to demonstrate the relative intensity and localization of protein nitration in OBs of 1.5-, 6-, and 20-month-old mice. Total protein carbonylation was significantly greater in OBs of 20- vs. 1.5-month-old mice. Aldolase 1 (ALDO1) showed significantly more carbonylation in OBs from 20- vs. 1.5-month-old mice; heat shock protein 9A and dihydropyrimidinase-like 2 showed significantly less. Several previously investigated proteins were also carbonylated, including ferritin heavy chain (FTH). Nitration, identified by 3-nitrotyrosine immunoreactivity, was least abundant at 1.5 months, intermediate at 6 months, and greatest at 20 months and was localized primarily in blood vessels. Proteins that were specific targets of oxidation were also localized: ALDO1 in astrocytes of the granule cell layer and FTH in mitral/tufted cells. These results indicate that specific carbonylated proteins, including those in astrocytes and mitral/tufted neurons, and nitrated proteins in the vasculature are molecular substrates of age-related olfactory dysfunction.

Aging↗

PubMed2006

Quantitative multiplexed profiling of cellular signaling networks using phosphotyrosine-specific DNA-tagged SH2 domains.

Deciphering global signaling networks is of great importance for the detailed understanding of cellular signaling processes controlling many important biological functions. Among signaling processes, tyrosine phosphorylation has a central role. At present, adequate techniques for the global characterization of the tyrosine phosphoproteome are lacking, particularly for the analysis of small amounts of protein. By combining the power of PCR amplification with the unique properties of Src homology region 2 (SH2) domains to specifically recognize tyrosine-phosphorylated proteins, we developed a new proteomic approach, termed oligonucleotide-tagged multiplex assay (OTM). For OTM, multiple SH2 domains are labeled by domain-specific oligonucleotide tags, applied as probes to complex protein mixtures in a multiplex reaction and phosphotyrosine-specific interactions are quantified by PCR. Using OTM we reproducibly quantified differential states of tyrosine phosphorylation with high sensitivity and specificity in small amounts of whole cellular extracts as demonstrated for various tumor cell lines and human leukemia samples.

3T3 Cells↗

PubMed2006

Enrichment of integral membrane proteins from small amounts of brain tissue.

Subcellular fractionation represents an essential technique for functional proteome analysis. Recently, we provided a subcellular fractionation protocol for minute amounts of tissue that yielded a nuclear fraction, a membrane and organelle fraction, and a cytosolic fraction. In the current study, we attempted to improve the protocol for the isolation of integral membrane proteins, as these are particularly important for brain function. In the membrane and organelle fraction, we increased the yield of membranes and organelles by about 50% by introducing a single re-extraction step. We then tested two protocols towards their capacity to enrich membrane proteins present in the membrane and organelle fraction. One protocol is based on sequential solubilization using subsequent increases of chaotropic conditions such as urea, thereby partitioning hydrophobic proteins from hydrophilic ones. The alternative protocol applies high-salt and high-pH washes to remove non-membrane proteins. The enrichment of membrane proteins by these procedures, as compared to the original membrane and organelle fraction, was evaluated by 16-BAC-SDS-PAGE followed by mass spectrometry of randomly selected spots. In the original membrane and organelle fraction, 7 of 50 (14%) identified proteins represented integral membrane proteins, and 15 (30%) were peripheral membrane proteins. In the urea-soluble fraction, 4 of 33 (12%) identified proteins represented integral membrane proteins, and 10 (30%) were peripheral membrane proteins. In the high-salt/high-pH resistant sediment, 12 of 45 (27%) identified proteins were integral membrane proteins and 13 (29%) represented peripheral membrane proteins. During the analysis, several proteins involved in neuroexocytosis were detected, including syntaxin, NSF, and Rab3-interaction protein 2. Taken together, differential centrifugation in combination with high-salt and high-pH washes resulted in the highest enrichment of integral membrane proteins and, therefore, represents an adequate technique for region-specific profiling of membrane proteins in the brain.

Animals↗

PubMed2006

Proteomic analysis of the allograft response.

BACKGROUND: As transcriptional profiling techniques appear to provide a useful means to evaluate the allograft response, we have now initiated an attempt to use proteomics to examine the allograft response. METHODS: To this end, we have evaluated the use of Protein Chip technology, coupled with bioinformatics analysis towards discovery of allograft response biomarkers in a mouse skin transplant model. To compare samples obtained from acutely rejecting allograft recipients at days 7, 9, and 21, we treated one group with a potent antirejection regimen. Mean survival time in the fully MHC-mismatched skin graft model using this protocol is more than 100 days. We also studied recipients of nontreated syngenetic grafts. We applied Protein Chip technology toward discovery of allograft response markers in this model. RESULTS: At days 7 and 9, before the clinical appearance of rejection at day 10, several protein biomarker candidates were detected, based on their molecular mass that clearly differentiated between rejection and the nonrejection groups. CONCLUSIONS: Protein profiling of serum as a means to characterize the allografts response of a given host deserves further testing in clinical studies.

Abatacept↗

PubMed2006

Proteome analysis of cultured fibroblasts from type 1 diabetic patients and normal subjects.

CONTEXT: Protein profiling of diabetic tissues could provide useful biomarkers for early diagnosis, therapeutic targets, and disease response markers. Cultured fibroblasts are a useful in vitro model for proteome analysis and study of the molecular mechanisms involved in diabetes. OBJECTIVE: The objective of the study was to isolate and characterize the proteins of cultured fibroblasts, obtained by skin biopsy, from long-term type 1 diabetic patients without complications and age- and sex-matched normal subjects as controls. DESIGN: Proteins were separated by two-dimensional electrophoresis (2-DE), and the gel images were qualitatively and quantitatively analyzed. Protein identification was performed by matrix-assisted laser desorption/ionization mass spectrometry. RESULTS: Reproducible protein maps of fibroblasts from diabetic and healthy subjects were obtained. A total of 125 protein spots were isolated and identified, among them 27 proteins not previously reported in published human fibroblast 2-DE maps, including 20 proteins never reported previously in the literature in human skin fibroblasts. Quantitative analyses revealed six protein spots differentially expressed in the fibroblasts from the diabetic vs. the control subjects (P < 0.05), representing glycolytic enzymes and structural proteins. An increase of triosephosphate I isomerase of two splice isoforms of pyruvate kinase and alpha-actinin 4 and a decrease of tubulin-beta2 and splice isoform 2 of tropomyosin beta-chain were detected. CONCLUSIONS: We generated 2-DE reference maps of the proteome of human skin fibroblasts from both normal and uncomplicated type 1 diabetic patients. Differences in glycolytic enzymes and structural proteins were found. The functional implications of the identified proteins are discussed.

Adult↗

PubMed2002

Studies of potential cerebrospinal fluid molecular markers for Alzheimer's disease.

There is a need for a reliable, molecular-based ante mortem diagnostic test for Alzheimer's disease (AD). In this study, we examined the use of two-dimensional protein electrophoresis for generating molecular barcodes which may be useful for the clinical differentiation of AD patients from normals. We compared cerebrospinal fluid samples taken from AD patients with confirmed post mortem pathology to comparable specimens from normal volunteers. Using canonical correlation analysis, a panel of nine molecular markers were identified which segregated diseased cases from normal controls. Using the scaled volume image analysis variable, a principal factor analysis was also used to distinguish normal from AD spinal fluid, based on molecular markers identified using a heuristic clustering algorithm. The use of panels of molecular markers derived from proteomic analysis may offer the best prospect for developing molecular diagnostic tests for complex neurodegenerative disorders such as AD.

Adult↗

PubMed2004

Perilipin A mediates the reversible binding of CGI-58 to lipid droplets in 3T3-L1 adipocytes.

Perilipins, the major structural proteins coating the surfaces of mature lipid droplets of adipocytes, play an important role in the regulation of triacylglycerol storage and hydrolysis. We have used proteomic analysis to identify CGI-58, a member of the alpha/beta-hydrolase fold family of enzymes, as a component of lipid droplets of 3T3-L1 adipocytes. CGI-58 mRNA is highly expressed in adipose tissue and testes, tissues that also express perilipins, and at lower levels in liver, skin, kidney, and heart. Both endogenous CGI-58 and an ectopic CGI-58-GFP chimera show diffuse cytoplasmic localization in 3T3-L1 preadipocytes, but localize almost exclusively to the surfaces of lipid droplets in differentiated 3T3-L1 adipocytes. The localization of endogenous CGI-58 was investigated in 3T3-L1 cells stably expressing mutated forms of perilipin using microscopy. CGI-58 binds to lipid droplets coated with perilipin A or mutated forms of perilipin with an intact C-terminal sequence from amino acid 382 to 429, but not to lipid droplets coated with perilipin B or mutated perilipin A lacking this sequence. Immunoprecipitation studies confirmed these findings, but also showed co-precipitation of perilipin B and CGI-58. Remarkably, activation of cAMP-dependent protein kinase by the incubation of 3T3-L1 adipocytes with isoproterenol and isobutylmethylxanthine disperses CGI-58 from the surfaces of lipid droplets to a cytoplasmic distribution. This shift in subcellular localization can be reversed by the addition of propanolol to the culture medium. Thus, CGI-58 binds to perilipin A-coated lipid droplets in a manner that is dependent upon the metabolic status of the adipocyte and the activity of cAMP-dependent protein kinase.

1-Acylglycerol-3-Phosphate O-Acyltransferase↗

PubMed2004

Proteomics in the chicken: tools for understanding immune responses to avian diseases.

The entire chicken genome sequence will be available by the time this review is in press. Chickens will be the first production animal species to enter the "postgenomic era." This fundamental structural genomics achievement allows, for the first time, complete functional genomics approaches for understanding the molecular basis of chicken normo- and pathophysiology. The functional genomics paradigm, which contrasts with classical functional genetic investigations of one gene (or few) in isolation, is the systematic holistic genetic analyses of biological systems in defined contexts. Context-dependent gene interactions are the fundamental mechanics of all life. Functional genomics uses high-throughput large-scale experimental methods combined with statistical and computational analyses. Projects with expressed sequence tags in chickens have already allowed the creation of cDNA microarrays for large-scale context-dependant mRNA analysis (transcriptomics). However, proteins are the functional units of almost all biological processes, and protein expression very often bears no correlation to mRNA expression. Proteomics, a discipline within functional genomics, is the context-defined analysis of complete complements of proteins. Proteomics bridges the "sequence-to-phenotype gap;" it complements structural and other functional genomics approaches. Proteomics requires high capital investment but has ubiquitous biological applications. Although currently the fastest-growing human biomedical discipline, new paradigms may need to be established for production animal proteomics research. The prospective promise and potential pitfalls of using proteomics approaches to improve poultry pathogen control will be specifically highlighted. The first stage of our recently established proteomics program is global protein profiling to identify differentially expressed proteins in the context of the commercially important pathogens. Our trials and tribulations in establishing our proteomics program, as well some of our initial data to understand chicken immune system function, will be discussed.

Animals↗

PubMed2003

High-throughput proteomics and protein biomarker discovery in an experimental model of inflammatory hyperalgesia: effects of nimesulide.

OBJECTIVE: The goal of this study was to derive a systematic approach for the identification of protein species profiles that selectively identify subgroups of similar but distinct pain models and correlate with the therapeutic efficacy (or lack thereof) of drug intervention. METHODS: Using high-throughput surface-enhanced laser desorption ionization (SELDI) mass spectrometry proteomic technology based on ProteinChip arrays, we conducted a comparative analysis of the profile of protein expression in cerebral spinal fluid (CSF) from rats exposed to either injection of complete Freund's adjuvant (CFA) [a model of inflammatory pain] or chronic constriction injury (CCI) [a model of neuropathic pain]. The CFA model was then further studied for the effects of treatment with the NSAID, nimesulide (a preferential cyclo-oxygenase [COX]-2 inhibitor). RESULTS: Among other observations, we found that the content of two metal (copper) binding protein species (2.9 and 3.2kDa) and three anionic protein species (4.0, 6.9 and 8.2kDa) were increased in the CSF of rats with inflammatory pain in a time-dependent fashion, at 7 and 14 days after CFA injection. These changes were highly selective for the CFA model, as no detectable increase in these protein biomarkers was found in the CCI neuropathic pain model. Further, we found that most of the changes in the biomarker protein species induced by the inflammatory pain were prevented by treatment with nimesulide and correlated with the antihyperalgesic effect of this drug. CONCLUSION: This study demonstrates that CSF biomarker profiles, as detected by SELDI technology, can consistently and reproducibly differentiate inflammatory from neuropathic pain, and reflect the analgesic action produced by the preferential COX-2 inhibitor, nimesulide. The characterisation and identification of these biomarkers will provide invaluable insight into the pathophysiology of pain mechanisms, in addition to further understanding of the value of nimesulide in the treatment of inflammatory pain.

Analysis of Variance↗

PubMed2006

Methanococcus aeolicus sp. nov., a mesophilic, methanogenic archaeon from shallow and deep marine sediments.

Three strains of CO(2)-reducing methanogens were isolated from marine sediments. Strain PL-15/H(P) was isolated from marine sediments of the Lipari Islands, near Sicily and the other two strains, Nankai-2 and Nankai-3(T), were isolated from deep marine sediments of the Nankai Trough, about 50 km from the coast of Japan. Analysis of the cellular proteins and 16S rRNA gene sequences indicated that these three strains represented a single novel species that formed a deep branch of the mesophilic methanococci. Phylogenetic analysis indicated that the three strains were most closely related to Methanothermococcus okinawensis (95 % 16S rRNA gene sequence similarity). However, strains PL-15/H(P), Nankai-2 and Nankai-3(T) grew at temperatures that were more similar to those of recognized species within the genus Methanococcus. Strain Nankai-3(T) grew fastest at 46 degrees C. Results of physiological and biochemical tests allowed the genotypic and phenotypic differentiation of strains PL-15/H(P), Nankai-2 and Nankai-3(T) from closely related species. The name Methanococcus aeolicus sp. nov. is proposed, with strain Nankai-3(T) (=OCM 812(T)=DSM 17508(T)) as the type strain.

Archaeal Proteins↗

PubMedDate not supplied

Bovine dilated cardiomyopathy: proteomic analysis of an animal model of human dilated cardiomyopathy.

Bovine hereditary dilated cardiomyopathy (bCMP) is endemic in Switzerland and hearts from diseased animals display important clinical and biochemical similarities to human DCM. Recent research has identified at least one protein (myoglobin) to be significantly reduced in bovine DCM. Using a proteomic approach, we have separated over 1125 protein species from bovine ventricular tissue. Gel analysis and protein characterisation have identified a number of proteins whose abundance is significantly altered in bovine DCM. Twenty-four proteins are of decreased abundance in diseased tissue, whilst 11 proteins are of increased abundance in the diseased state. A combination of amino acid compositional analysis, peptide mass profiling, N-terminal microsequencing and MultiIdent (http://www.expasy.ch/sprot/multiident. html) has been employed in order to elucidate the identities of the differentially expressed proteins. Using these techniques we have currently determined the identity of 12 of the 35 altered proteins. We have also detected three proteins that are differentially expressed in genotypically diseased but phenotypically normal animals, identifying a possible mechanism for the onset of the disease. The possibility that inappropriate ubiquination of proteins plays an important role in the disease is discussed. A database of bovine proteins is currently being established. The identity of the proteins affected, together with a comparison of the human and bovine expression patterns, is displayed.

Animals↗

PubMed2024

Proteome analysis provides insights into sex differences in Holothuria Scabra.

Sex-determining mechanism is still ambiguous for sea cucumber Holothuria scabra which only manifests gonochorism in gonad. In this study, proteomic analysis was employed to delineate sex-related proteins and genes in gonads of H. scabra, subsequently validated through Quantitative real-time polymerase chain reaction (qRT-PCR). A total of 5,313 proteins were identified via proteome sequencing. Among these, 817 proteins exhibited expression in both the ovary and testis, with 445 proteins displaying up-regulation and 372 proteins showing down-regulation (ovary vs testis). Furthermore, 136 and 69 proteins were identified as ovary-specific and testis-specific Differentially Abundant Proteins (DAPs), respectively. And 9 DAP coding genes which play crucial role in ovary and testis were verified by qRT-PCR. Notably, 24 ovary-bias proteins enriched in ribosome pathway strongly indicated the crucial role of ribosome in ovary. This study serves to furnish novel evidence pertaining to sex differences in H. scabra.

Animals↗

PubMed2006

Comparative profiling of the mammalian mitochondrial proteome: multiple aconitase-2 isoforms including N-formylkynurenine modifications as part of a protein biomarker signature for reactive oxidative species.

The activity of mitochondria induces, as a byproduct, a variety of post-translational modifications in associated proteins, which have functional downstream consequences for processes such as apoptosis, autophagy, and plasticity; e.g., reactive oxygen species (ROS), which induce N-formyl-kynurenine from oxidized tryptophans in certain mitochondrial proteins which are localized in close spatial proximity to their source. This type of fast molecular changes has profound influence on cell death and survival with implications in a number of pathologies. The quantitative and differential analysis of bovine heart mitochondria by four 2D-PAGE methods, including 2D-PAGE with high-resolution IEF as first dimension, revealed that due to limited resolution, those methods employing blue native-, tricine-urea-, and 16-BAC-PAGE as the first dimension are less applicable for the differential quantitative analysis of redundant protein spots which might give insight into post-translational modifications that are relevant in age- and stress-related changes. Moreover, 2D-PAGE with high resolution IEF was able to resolve a surprisingly large number of membrane proteins from mitochondrial preparations. For aconitase-2, an enzyme playing an important role in mitochondrial aging, a more thorough molecular analysis of all separable isoforms was performed, leading to the identification of two particular N-formylkynurenine modifications. Next to protein redundancy, native protein-protein interactions, with the potential of relating certain post-translational modification patterns to distinct oligomeric states, e.g., oxidative phosphorylation super complexes, might provide novel and (patho-) physiologically relevant information. Among proteins identified, 14 new proteins (GenBank entries), previously not associated with mitochondria, were found.

Aconitate Hydratase↗