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PubMed2002

A proteomic approach to identify developmentally regulated proteins in Leishmania infantum.

We used comparative two-dimensional gel electrophoresis (2-DE) and mass spectrometry methodologies to highlight and identify proteins that are differentially expressed in the intracellular stage of the parasite Leishmania donovani infantum, a causative agent of visceral leishmaniasis. During its digenetic life cycle, Leishmania alternates between the alimentary tract of the sandfly vector as an extracellular promastigote and the acidic phagolysosomes of macrophage cells as an intracellular amastigote. Proteins differentially expressed in the intracellular form of the parasite are thought to be important for intracellular survival and pathogenesis. We used narrow pH range strips for isoelectric focusing to resolve soluble proteins of both developmental stages of L. infantum. More than 62 proteins differentially expressed in amastigotes were detected among approximately 2000 protein spots resolved by 2-DE. A quadrupole time-of-flight analysis of few selected protein spots, specifically expressed in the amastigote stage, permitted the identification of two proteins, part of the energetic metabolism pathways, the isocitrate dehydrogenase and the glycolytic enzyme triosephosphate isomerase. The kinetic parameters of these two enzymes were measured in both developmental stages of the parasite and their activity was indeed found to be higher in amastigotes. These findings bring a new insight in our understanding of metabolic and energy requirements of the intracellular form of Leishmania. Comparative analysis of the proteome of both developmental stages of the protozoan parasite Leishmania should permit the identification of protein candidates for the development of vaccines and new drugs.

Amino Acid Sequence↗
PubMed2005

Gel-based versus gel-free proteomics: a review.

With the sequencing of the genome of over 150 organisms, the field of biology has been revolutionised. Instead of studying one gene or protein at the time, it is now possible to study the effect of physiological or pathological changes on the expression of all genes or proteins in the organism. Proteomics aims at the simultaneous analysis of all proteins expressed by a cell, tissue or organism in a specific physiological condition. Because proteins are the effector molecules in all organisms, it is evident that changes in the physiological condition of an organism will be reflected by changes in protein expression and/or processing. Since the formulation of the concept of proteomics in the mid 90's proteomics has relied heavily on 2 dimensional gel electrophoresis (2DGE) for the separation and visualization of proteins. 2DGE, however, has a number of inherent drawbacks. 2DGE is costly, fairly insensitive to low copy proteins and cannot be used for the entire proteome. Therefore, over the years, several gel-free proteomics techniques have been developed to either fill the gaps left by 2DGE or to entirely abolish the gel based techniques. This review summarizes the most important gel-free and gel-based proteomics techniques and compares their advantages and drawbacks.

Electrophoresis, Polyacrylamide Gel↗
PubMedDate not supplied

Liquid chromatography MALDI MS/MS for membrane proteome analysis.

Membrane proteins play critical roles in many biological functions and are often the molecular targets for drug discovery. However, their analysis presents a special challenge largely due to their highly hydrophobic nature. We present a surfactant-aided shotgun proteomics approach for membrane proteome analysis. In this approach, membrane proteins were solubilized and digested in the presence of SDS followed by newly developed auto-offline liquid chromatography/matrix-assisted laser desorption ionization (LC/MALDI) tandem MS analysis. Because of high tolerance of MALDI to SDS, one-dimensional (1D) LC separation can be combined with MALDI for direct analysis of protein digests containing SDS, without the need for extensive sample cleanup. In addition, the heated droplet interface used in LC/MALDI can work with high flow LC separations, allowing a relatively large amount of protein digest to be used for 1D LC/MALDI which facilitates the detection of low abundance proteins. The proteome identification results obtained by LC/MALDI are compared to the gel electrophoresis/MS method as well as the shotgun proteomics method using 2D LC/electrospray ionization MS. It is demonstrated that, while LC/MALDI provides more extensive proteome coverage compared to the other two methods, these three methods are complementary to each other and a combination of these methods should provide a more comprehensive membrane proteome analysis.

Chromatography, High Pressure Liquid↗
PubMed2004

Complexity analysis of yeast proteome network.

Topological and compositional complexity of protein-protein networks is assessed in a variety of ways making use of graph theory and information theory. The methodology used is borrowed from mathematical chemistry and includes complexity descriptors such as substructure count, overall connectivity, walk count, and information on various vertex distributions. The approach is applied to the (incomplete) proteome of Saccharomyces cerevisiae containing 232 protein complexes of a total of 1,440 proteins. The proteome network and each of its nine functional subsets of protein complexes are disconnected graphs, containing a number of noninteracting species and a major component. A weighted edge between two vertices in these graphs stands for the number of shared proteins between the respective complexes. The major component is a highly connected, 'small-world' network, in which the average vertex distance between protein complexes does not exceed 2.2 (2.4 for the entire proteome), whereas the maximum distance does not exceed 4 (or 5 for the proteome). The vertex degree distribution in the major proteome component with 199 complexes follows the power law P(k) approximately k(-gamma), with gamma approximately = 1.7. The analysis of the functional organization of the yeast proteome has shown that, for any pair of biological functions, there always exist many proteins that can perform both functions. The potential application of the quantitative proteome descriptors discussed includes quantitative relationships between the structure and biological action of dynamic protein complexes in changing environment, identification of targets for markers/drugs, as well as system analysis and comparative studies of proteomes.

Fungal Proteins↗
PubMed2006

Dynamics of Arabidopsis thaliana soluble proteome in response to different nutrient culture conditions.

In an effort to determine the best extraction procedure compatible with the high-reproducible 2-DE, different methods of soluble protein extraction from Arabidopsis cell culture suspensions grown in Gamborg B5 medium were tested. A reference 2-DE map was established for this soluble extract revealing 1184 spots. The most abundant protein spots were excised, trypsin-digested, and mass spectra obtained via MALDI-TOF and/or LC coupled to ESI-MS. Three hundred and thirty one proteins were identified and their functions were defined based on sequence comparisons and classified in different protein families. In order to analyze the impact of culture medium on the Arabidopsis proteome, we performed the 2-DE map from Arabidopsis cell suspensions cultured in another growth medium Murashige and Skoog (M-S) and 327 major spots were identified. Using PDQuest imaging analysis, significant increases in the amount of several housekeeping enzymes, stress/defense proteins, and heat shock proteins were found in M-S medium. Modified expression of certain proteins and detection of new isoforms involved in nitrate assimilation, nitrogen, and sulfur metabolism were also observed in the M-S medium. This study provides the first 2-DE maps of the soluble proteome of Arabidopsis cell suspensions. The comparative analysis of the Arabidopsis proteome in respect to different nutrient supplies shows that the culture medium may significantly influence the expression pattern of major soluble proteins in Arabidopsis cells. This work also constitutes an important step for further proteomic analysis concerning cell responses to abiotic or biotic stresses.

Arabidopsis↗
PubMed2006

Tissue heterogeneity of the mammalian mitochondrial proteome.

The functionality of the mitochondrion is primarily determined by nuclear encoded proteins. The mitochondrial functional requirements of different tissues vary from a significant biosynthetic role (liver) to a primarily energy metabolism-oriented organelle (heart). The purpose of this study was to compare the mitochondrial proteome from four different tissues of the rat, brain, liver, heart, and kidney, to provide insight into the extent of mitochondrial heterogeneity and to further characterize the overall mitochondrial proteome. Mitochondria were isolated, solubilized, digested, and subjected to quantitative liquid chromatography-mass spectroscopy. Of the 16,950 distinct peptides detected, 8,045 proteins were identified. High-confidence identification threshold was reached by 1,162 peptides, which were further analyzed. Of these 1,162 proteins, 1,149 were significantly different in content (P and q values < 0.05) between at least 2 tissues, whereas 13 were not significantly different between any tissues. Confirmation of the mitochondrial origin of proteins was determined from the literature or via NH(2)-terminal mitochondrial localization signals. With these criteria, 382 proteins in the significantly different groups were confirmed to be mitochondrial, and 493 could not be confirmed to be mitochondrial but were not definitively localized elsewhere in the cell. A total of 145 proteins were assigned to the rat mitochondrial proteome for the first time via their NH(2)-terminal mitochondrial localization signals. Among the proteins that were not significantly different between tissues, three were confirmed to be mitochondrial. Most notable of the significantly different proteins were histone family proteins and several structural proteins, including tubulin and intermediate filaments. The mitochondrial proteome from each tissue had very specific characteristics indicative of different functional emphasis. These data confirm the notion that mitochondria are tuned by the nucleus for specific functions in different tissues.

Amino Acid Sequence↗
PubMed2004

Application of proteomics in phylogenetic and evolutionary studies.

There are few papers that deal specifically with evolutionary studies and proteomics. However, applying proteomics to these studies promises to open new perspectives apropos the construction of phylogenetic trees and the detection of evolutionary changes. Principles and methods of phylogenetic systematics could be used to compare and evaluate proteomes. This would permit the detection and characterization of specific proteins that have evolutionary value in defining monophyly, paraphyly, and polyphyly.

Algorithms↗
PubMed2006

Proteomic analysis of pathogenic bacterium Vibrio vulnificus.

In this study we have constructed a proteome reference map of the pathogenic bacterium Vibrio vulnificus. From the reference map, we identified several virulence-related proteins, such as ToxR and ToxS, as well as numerous proteins involved in diverse cellular functions. To search for additional virulence-related proteins, we compared the whole proteomes from the wild-type and toxR mutant of V. vulnificus and found that several proteins were up- or down-regulated in the toxR mutant. We suggest that these differentially regulated proteins whose expression is coordinately controlled by a virulence regulator ToxR, some of which are already implicated in virulence, play roles in the pathogenesis of V. vulnificus.

Bacterial Proteins↗
PubMed2002

Development of improved cell lysis, solubilization and imaging approaches for proteomic analyses.

Analysis of complex biochemical processes at the level of the proteome requires methods that quantitatively solubilize cytosolic and membrane bound proteins yet are compatible with isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In addition, it is often necessary to employ several highly sensitive detection methods to identify key proteins that are modified or exhibit a change in expression levels in response to a given experimental stimulus or condition. Methods were developed that efficiently extract tissues or lyse cultured cells and quantitatively solubilize proteins in a single step without the need to shear nucleic acids. These approaches utilize urea, thiourea, a mixture of detergents, low levels of an ampholyte blend, reductant and a combination of alcohols. To aid in the detection of low abundance proteins and the accurate identification of specific proteins of interest in these samples, two approaches were pursued. In one, proteins are transferred from two-dimensional (2-D) gels to blot membranes. Proteins are then detected by staining with SYPRO Ruby and the resulting 2-D protein pattern is captured using a charge-coupled device (CCD) camera. The blots are then probed with antibodies directed against the protein(s) or functionalities of interest. The resulting chemiluminescent blot image is also generated with the CCD camera and the fluorescent SYPRO Ruby image is recaptured again without moving the membrane. It is thereby possible to generate a direct image overlay of the blot pattern on that of the stained protein pattern. This approach significantly aids in the accurate identification of the dye-stained protein that is detected by the specific antibody. In addition to detecting protein post-gel transfer, a second approach utilizes protein samples labeled with fluorescent dyes prior to 2-D electrophoresis in an effort to increase the sensitivity of protein detection and to facilitate protein quantitation. It is also possible to stain the blots with different dyes and overlay these images as well. Using these approaches, it is possible to perform more rapid and accurate comparative analyses and proteomic, post-gel characterization of proteins of interest than using comparative image analysis of multiple gels.

Blotting, Western↗
PubMed2004

Shaping the mitochondrial proteome.

Mitochondria are eukaryotic organelles that originated from a single bacterial endosymbiosis some 2 billion years ago. The transition from the ancestral endosymbiont to the modern mitochondrion has been accompanied by major changes in its protein content, the so-called proteome. These changes included complete loss of some bacterial pathways, amelioration of others and gain of completely new complexes of eukaryotic origin such as the ATP/ADP translocase and most of the mitochondrial protein import machinery. This renewal of proteins has been so extensive that only 14-16% of modern mitochondrial proteome has an origin that can be traced back to the bacterial endosymbiont. The rest consists of proteins of diverse origin that were eventually recruited to function in the organelle. This shaping of the proteome content reflects the transformation of mitochondria into a highly specialized organelle that, besides ATP production, comprises a variety of functions within the eukaryotic metabolism. Here we review recent advances in the fields of comparative genomics and proteomics that are throwing light on the origin and evolution of the mitochondrial proteome.

Animals↗
PubMed2005

1-DE MS and 2-D LC-MS analysis of the mouse bronchoalveolar lavage proteome.

Bronchoalveolar lavage fluid (BALF) is a complex mixture of proteins, which represents a unique clinically useful sampling of the lower respiratory tract. Many proteomic technologies can be used to characterize complex biological mixtures; however, it is not yet clear which technology(s) provide more information regarding the number of proteins identified and sequence coverage. In this study, we initially compared two common proteomic approaches, 2-D LC microESI MS/MS and 1-DE followed by gel slice digestion, peptide extraction and peptide identification by MS in characterization of the mouse BALF proteome; secondly, we identified 297 unique proteins from the mouse BALF proteome, greatly expanded the BALF proteome by about threefold regardless of species.

Amino Acid Sequence↗
PubMed2006

Proteomic analysis of cerebrospinal fluid discriminates malignant and nonmalignant disease of the central nervous system and identifies specific protein markers.

CNS diseases are often accompanied by changes in the protein composition of cerebrospinal fluid (CSF). SELDI-TOF-MS provides an approach for identifying specific protein markers of disease in biological fluids. We compared the CSF proteomes from patients with neoplastic and reactive/inflammatory CNS diseases to identify potential biomarkers. SELDI-TOF-MS was performed on CSF derived from lumbar puncture of 32 patients, including 10 with CNS malignancies, 12 with inflammatory or reactive conditions, and 10 with unknown CNS disease. Using the SAX-2 (strong anionic exchange) chip, we uncovered three conserved protein peak ranges within each disease category. For neoplastic diseases, we identified conserved peaks at 7.5-8.0 kDa (9/10 samples), 15.1-15.9 kDa (8/10 samples), and 30.0-32.0 kDa (5/10 samples). In reactive/inflammatory diseases, conserved peaks were found at 6.7-7.1 kDa (10/12 samples), 11.5-11.9 kDa (12/12 samples), and 13.3-13.7 kDa (9/12 samples). A protein from the 30.0 to 32.0 kDa peak range found in neoplastic CSF was identified by MALDI analysis as carbonic anhydrase, a protein overexpressed in many malignancies including high-grade gliomas. Similarly, cystatin C was identified in the 13.3-13.7 kDa peak range in non-neoplastic CSF and was most prominent in inflammatory conditions. Our approach provides a rational basis for identifying biomarkers that could be used for detection, diagnosis, and monitoring of CNS diseases.

Adult↗
PubMed2004

The urinary proteome in Fanconi syndrome implies specificity in the reabsorption of proteins by renal proximal tubule cells.

Polypeptides present in the glomerular filtrate are almost completely reabsorbed in the first segment of the proximal tubule by receptor-mediated endocytosis; in renal Fanconi syndrome (FS), there is failure to reabsorb many of these polypeptides. We have compared the urinary proteomes in patients with Dent's disease (due to a CLC5 mutation), a form of FS, with normal subjects using three different proteomic methods. No differences in the levels of several plasma proteins were detected when standardized to total protein amounts. In contrast, several vitamin and prosthetic group carrier proteins were found in higher amounts in Dent's urine (with respect to total protein). Similarly, complement components, apolipoproteins, and some cytokines represented a larger proportion of the Dent's urinary proteome, suggesting that such proteins are reabsorbed more efficiently than other classes of proteins. Conversely, proteins of renal origin were found in proportionately higher amounts in normal urine. Thus the uptake of filtered vitamins, which are normally bound to their respective carrier proteins to prevent urinary losses, seems a key function of the proximal tubule; in addition, this nephron segment may also play a critical role in reabsorbing potentially cytotoxic polypeptides of plasma origin, preventing them from acting at more distal nephron sites.

Adolescent↗
PubMed2006

Comparative model of EutB from coenzyme B12-dependent ethanolamine ammonia-lyase reveals a beta8alpha8, TIM-barrel fold and radical catalytic site structural features.

The structure of the EutB protein from Salmonella typhimurium, which contains the active site of the coenzyme B12 (adenosylcobalamin)-dependent enzyme, ethanolamine ammonia-lyase, has been predicted by using structural proteomics techniques of comparative modelling. The 453-residue EutB protein displays no significant sequence identity with proteins of known structure. Therefore, secondary structure prediction and fold recognition algorithms were used to identify templates. Multiple three-dimensional template matching (threading) servers identified predominantly beta8alpha8, TIM-barrel proteins, and in particular, the large subunits of diol dehydratase (PDB: 1eex:A, 1dio:A) and glycerol dehydratase (PDB: 1mmf:A), as templates. Consistent with this identification, the dehydratases are, like ethanolamine ammonia-lyase, Class II coenzyme B12-dependent enzymes. Model building was performed by using MODELLER. Models were evaluated by using different programs, including PROCHECK and VERIFY3D. The results identify a beta8alpha8, TIM-barrel fold for EutB. The beta8alpha8, TIM-barrel fold is consistent with a central role of the alpha/beta-barrel structures in radical catalysis conducted by the coenzyme B12- and S-adenosylmethionine-dependent (radical SAM) enzyme superfamilies. The EutB model and multiple sequence alignment among ethanolamine ammonia-lyase, diol dehydratase, and glycerol dehydratase from different species reveal the following protein structural features: (1) a "cap" loop segment that closes the N-terminal region of the barrel, (2) a common cobalamin cofactor binding topography at the C-terminal region of the barrel, and (3) a beta-barrel-internal guanidinium group from EutB R160 that overlaps the position of the active-site potassium ion found in the dehydratases. R160 is proposed to have a role in substrate binding and radical catalysis.

Amino Acid Sequence↗
PubMed2002

A question of size: the eukaryotic proteome and the problems in defining it.

We discuss the problems in defining the extent of the proteomes for completely sequenced eukaryotic organisms (i.e. the total number of protein-coding sequences), focusing on yeast, worm, fly and human. (i) Six years after completion of its genome sequence, the true size of the yeast proteome is still not defined. New small genes are still being discovered, and a large number of existing annotations are being called into question, with these questionable ORFs (qORFs) comprising up to one-fifth of the 'current' proteome. We discuss these in the context of an ideal genome-annotation strategy that considers the proteome as a rigorously defined subset of all possible coding sequences ('the orfome'). (ii) Despite the greater apparent complexity of the fly (more cells, more complex physiology, longer lifespan), the nematode worm appears to have more genes. To explain this, we compare the annotated proteomes of worm and fly, relating to both genome-annotation and genome evolution issues. (iii) The unexpectedly small size of the gene complement estimated for the complete human genome provoked much public debate about the nature of biological complexity. However, in the first instance, for the human genome, the relationship between gene number and proteome size is far from simple. We survey the current estimates for the numbers of human genes and, from this, we estimate a range for the size of the human proteome. The determination of this is substantially hampered by the unknown extent of the cohort of pseudogenes ('dead' genes), in combination with the prevalence of alternative splicing. (Further information relating to yeast is available at http://genecensus.org/yeast/orfome)

Animals↗
PubMed2006

An initial proteomic analysis of human preterm labor: placental membranes.

Human preterm labor (PL) is the single most significant problem in modern Obstetrics and Gynecology, affecting approximately 10% of pregnancies worldwide, constituting the leading cause of perinatal mortality and morbidity, and contributing significantly to chronic childhood disease. Currently, our molecular understanding of PL remains staggeringly inadequate to reliably diagnose or rationally intervene in PL events; several molecular alterations have been implicated in PL, but these have proven of limited value as diagnostic/prognostic markers. The majority of PL events remain spontaneous and unpredictable: critical care emergencies. Here, we apply functional proteomics to dissect molecular mechanisms of human PL. Human placental tissue was collected in clearly differentiated cases of preterm and term labor. Highly refined two-dimensional gel electrophoresis (2DE) was used for protein separation, coupled with automated differential gel image analysis to compare the resulting proteomic maps. For this initial study, only the most important protein differences were selected for further analysis, that is, proteins that were unique to one sample, and absent from the other, with 100% reproducibility across the sample population. In total, 11 such proteins were identified by tandem mass spectrometry, falling into three distinct functional classes: structural/cytoskeletal components, ER lumenal proteins with enzymatic or chaperone functions, and proteins with anticoagulant properties. These expression changes form the groundwork for further molecular investigation of this devastating medical condition. This approach therefore holds the potential not only to define the underlying molecular components, but also to identify novel diagnostic tools and targets for rational drug intervention.

Anticoagulants↗
PubMed2003

Proteome analysis of recombinant xylose-fermenting Saccharomyces cerevisiae.

Introduction of an active xylose utilization pathway into Saccharomyces cerevisiae, which does not naturally ferment pentose sugars, is likely to have a major impact on the overall cellular metabolism as the carbon introduced to the cells will now flow through the pentose phosphate pathway. The metabolic responses in the recombinant xylose-fermenting S. cerevisiae were studied at the proteome level by comparative two-dimensional gel electrophoresis of cellular proteins within a pH range of 3-10. Glucose-limited chemostat cultivations and corresponding chemostat cultivations performed in media containing xylose as the major carbon source were compared. The cultivations were studied in aerobic and anaerobic metabolic steady states and in addition at time points 5, 30 and 60 min after the switch-off of oxygen supply. We identified 22 proteins having a significant abundance difference on xylose compared to glucose, and 12 proteins that responded to change from aerobic to anaerobic conditions on both carbon sources. On xylose in all conditions studied, major changes were seen in the abundance of alcohol dehydrogenase 2 (Adh2p), acetaldehyde dehydrogenases 4 and 6 (Ald4p and Ald6p), and DL-glycerol 3-phosphatase (Gpp1p). Our results give indications of altered metabolic fluxes especially in the acetate and glycerol pathways in cells growing on xylose compared to glucose.

Aerobiosis↗
PubMed2006

MKS1, encoding a component of the flagellar apparatus basal body proteome, is mutated in Meckel syndrome.

Meckel syndrome (MKS) is a severe fetal developmental disorder reported in most populations. The clinical hallmarks are occipital meningoencephalocele, cystic kidney dysplasia, fibrotic changes of the liver and polydactyly. Here we report the identification of a gene, MKS1, mutated in MKS families linked to 17q. Mks1 expression in mouse embryos, as determined by in situ hybridization, agrees well with the tissue phenotype of MKS. Comparative genomics and proteomics data implicate MKS1 in ciliary functions.

Abnormalities, Multiple↗