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Background Biological age may be estimated by proteomic aging clocks (PACs). Previous published PACs were constructed either in smaller studies or mainly in white individuals, and they used proteomic measures from only one-time point. In this study, we created de novo PACs and compared their performance to published PACs at 2 different time points in the Atherosclerosis Risk in Communities (ARIC) study of white and black participants (around 75% white and 25% black). Medthods and findings A total of 4,712 plasma proteins were measured using SomaScan in blood samples collected in 1990 to 1992 from 11,761 midlife participants (aged 46 to 70 years) and in 2011 to 2013 from 5,183 late-life participants (aged 66 to 90 years). The de novo ARIC PACs were constructed by training them against chronological age using elastic net regression in two-thirds of healthy participants in midlife and late life and validated in the remaining one-third of healthy participants at the corresponding time point. We also computed 3 published PACs. We estimated age acceleration for each PAC as residuals after regressing each PAC on chronological age. We also calculated the change in age acceleration from midlife to late life. We examined the associations of age acceleration and change in age acceleration with mortality through 2019 from all-cause, cardiovascular disease (CVD), cancer, and lower respiratory disease (LRD) using Cox proportional hazards regression in participants (irrespective of health) after excluding the training set. The model was adjusted for chronological age, smoking, body mass index (BMI), and other confounders. We externally validated the midlife PAC using the Multi-Ethnic Study of Atherosclerosis (MESA) Exam 1 data. The ARIC PACs had a slightly stronger correlation with chronological age than published PACs in healthy participants at each time point. Associations with mortality were similar for the ARIC PACs and published PACs. For late-life and midlife age acceleration for the ARIC PACs, respectively, hazard ratios (HRs) per 1 standard deviation were 1.65 and 1.38 (both p < 0.001) for all-cause mortality, 1.37 and 1.20 (both p < 0.001) for CVD mortality, 1.21 (p = 0.028) and 1.04 (p = 0.280) for cancer mortality, and 1.68 and 1.36 (both p < 0.001) for LRD mortality. For the change in age acceleration, HRs for all-cause, CVD, and LRD mortality were comparable to the HRs for late-life age acceleration. The association between the change in age acceleration and cancer mortality was not significant. The external validation of the midlife PAC in MESA showed significant associations with mortality, as observed for midlife participants in ARIC. The main limitation is that our PACs were constructed in midlife and late-life participants. It is unknown whether these PACs could be applied to young individuals. Conclusions In this longitudinal study, we found that the ARIC PACs and published PACs were similarly associated with an increased risk of mortality. These findings suggested that PACs show promise as biomarkers of biological age. PACs may be serve as tools to predict mortality and evaluate the effect of anti-aging lifestyle and therapeutic interventions., Author(s): Shuo Wang 1,*, Zexi Rao 2, Rui Cao 2, Anne H. Blaes 3, Josef Coresh 4, Rajat Deo 5, Ruth Dubin 6, Corinne E. Joshu 7,8, Benoit Lehallier 9, [...]

Gigantopithecus blacki was a giant hominid that inhabited densely forested environments of Southeast Asia during the Pleistocene epoch.sup.1. Its evolutionary relationships to other great ape species, and the divergence of these species during the Middle and Late Miocene epoch (16-5.3 million years ago), remain unclear.sup.2,3. Hypotheses regarding the relationships between Gigantopithecus and extinct and extant hominids are wide ranging but difficult to substantiate because of its highly derived dentognathic morphology, the absence of cranial and post-cranial remains.sup.1,3-6, and the lack of independent molecular validation. We retrieved dental enamel proteome sequences from a 1.9-million-year-old G. blacki molar found in Chuifeng Cave, China.sup.7,8. The thermal age of these protein sequences is approximately five times greater than that of any previously published mammalian proteome or genome. We demonstrate that Gigantopithecus is a sister clade to orangutans (genus Pongo) with a common ancestor about 12-10 million years ago, implying that the divergence of Gigantopithecus from Pongo forms part of the Miocene radiation of great apes. In addition, we hypothesize that the expression of alpha-2-HS-glycoprotein, which has not been previously observed in enamel proteomes, had a role in the biomineralization of the thick enamel crowns that characterize the large molars in Gigantopithecus.sup.9,10. The survival of an Early Pleistocene dental enamel proteome in the subtropics further expands the scope of palaeoproteomic analysis into geographical areas and time periods previously considered incompatible with the preservation of substantial amounts of genetic information. The enamel proteome from a 1.9-million-year-old Gigantopithecus tooth shows that the Gigantopithecus and Pongo (orangutan) lineages diverged 12-10 million years ago., Author(s): Frido Welker [sup.1] , Jazmín Ramos-Madrigal [sup.1] , Martin Kuhlwilm [sup.2] , Wei Liao [sup.3] [sup.4] , Petra Gutenbrunner [sup.5] , Marc de Manuel [sup.2] , Diana Samodova [sup.6] [...]

To what extent genotypic differences translate to phenotypic variation remains a poorly understood issue of paramount importance for several cornerstone concepts of microbiology including the species definition. Here, we take advantage of the completed genomic sequences, expressed proteomic profiles, and physiological studies of 10 closely related Shewanella strains and species to provide quantitative insights into this issue. Our analyses revealed that, despite extensive horizontal gene transfer within these genomes, the genotypic and phenotypic similarities among the organisms were generally predictable from their evolutionary relatedness. The power of the predictions depended on the degree of ecological specialization of the organisms evaluated. Using the gradient of evolutionary relatedness formed by these genomes, we were able to partly isolate the effect of ecology from that of evolutionary divergence and to rank the different cellular functions in terms of their rates of evolution. Our ranking also revealed that whole-cell protein expression differences among these organisms, when the organisms were grown under identical conditions, were relatively larger than differences at the genome level, suggesting that similarity in gene regulation and expression should constitute another important parameter for (new) species description. Collectively, our results provide important new information toward beginning a systems-level understanding of bacterial species and genera. comparative genomics | evolution | proteomics | speciation | phenotype

A strictly host-dependent lifestyle has profound evolutionary consequences for bacterial genomes. Most prominent is a sometimes-dramatic amount of gene loss and genome reduction. Recently, highly reduced genomes from the co-resident intracellular symbionts of sharpshooters were shown to exhibit a striking level of metabolic interdependence. One symbiont, called Sulcia muelleri (Bacteroidetes), can produce eight of the 10 essential amino acids, despite having a genome of only 245 kb. The other, Baumannia cicadellinicola ([gamma]-Proteobacteria), can produce the remaining two essential amino acids as well as many vitamins. Cicadas also contain the symbiont Sulcia, but lack Baumannia and instead contain the co-resident symbiont Hodgkinia cicadicola ([alpha]-Proteobacteria). Here we report that, despite at least 200 million years of divergence, the two Sulcia genomes have nearly identical gene content and gene order. Additionally, we show that despite being phyiogenetically distant and drastically different in genome size and architecture, Hodgkinia and Baumannia have converged on gene sets conferring similar capabilities for essential amino acid biosynthesis, in both cases precisely complementary to the pathways conserved in Sulcia. In contrast, they have completely divergent capabilities for vitamin biosynthesis. Despite having the smallest gene set known in bacteria, Hodgkinia devotes at least 7% of its proteome to cobalamin (vitamin [B.sub.12]) biosynthesis, a significant metabolic burden. The presence of these genes can be explained by Hodgkinia's retention of the cobalamin-dependent version of methionine synthase instead of the cobalamin-independent version found in Baumannia, a situation that necessitates retention of cobalamin biosynthetic capabilities to make the essential amino acid methionine. cobalamin | genome reduction | genome sequencing | proteomics

In order to survive in the host and initiate infection, Salmonella enterica needs to undergo a transition between aerobic and anaerobic growth by modulating its central metabolic pathways. In this study, a comparative analysis of the proteome of S. enterica serovar Typhimurium grown in the presence or absence of oxygen was performed. The most prominent changes in expression were measured in a semiquantitative manner using difference in-gel electrophoresis (DIGE) to reveal the main protein factors involved in the adaptive response to anaerobiosis. A total of 38 proteins were found to be induced anaerobically, while 42 were repressed. The proteins of interest were in-gel digested with trypsin and identified by MALDI TOF mass spectrometry using peptide mass fingerprinting. In the absence of oxygen, many fermentative enzymes catalysing reactions in the mixed-acid or arginine fermentations were overexpressed. In addition, the enzyme fumarate reductase, which is known to provide an alternative electron acceptor for the respiratory chains in the absence of oxygen, was shown to be induced. Increases in expression of several glycolytic and pentose phosphate pathway enzymes, as well as two malic enzymes, were detected, suggesting important roles for these in anaerobic metabolism. Substantial decreases in expression were observed for a large number of periplasmic transport proteins. The majority of these are involved in the uptake of amino acids and peptides, but permeases transporting iron, thiosulphate, glucose/galactose, glycerol 3-phosphate and dicarboxylic acids were also repressed. Decreases in expression were also observed for a superoxide dismutase, ATP synthase, inositol monophosphatase, and several chaperone and hypothetical proteins. The changes were monitored in two different isolates, and despite their very similar expression patterns, some variability in the adaptive response to anaerobiosis was also observed.

[alpha]-Glucosidase I regulates trimming of the terminal [alpha]-l,2-glucose residue in the N-glycan processing pathway, which plays an important role in quality control systems in mammalian cells. Previously, we identified the gene encoding [alpha]-glucosidase I in the opportunistic human fungal pathogen Aspergillus fumigatus, namely Afcwh41. Deletion of the Afcwh41 gene results in a severe reduction of conidia formation, a temperature-sensitive deficiency of cell wall integrity, and abnormalities of polar growth and septation. An upregulation of the genes encoding Rho-type GTPases was also observed, which suggests activation of the cell wall integrity pathway in the mutant. Using 2D gel analysis, we revealed that the proteins involved in protein assembly, ubiquitin-mediated degradation and actin organization are altered in the [DELTA]Afcwh41 mutant. Evidence was obtained for a defect in the polarized localization of the actin cytoskeleton in the mutant. Our results suggest that blocking of the glucose trimming in A. fumigatus might induce accumulation of misfolded proteins in the endoplasmic reticulum; these misfolded proteins are probably required for cell wall synthesis and thus activate the cell wall integrity pathway, which then causes the abnormal polarity associated with the [DELTA]Afcwh41 mutant.

Mass spectrometry (MS)-based quantitative interaction proteomics has successfully elucidated specific protein--protein, DNA--protein, and small molecule--protein interactions. Here, we developed a gel-free, sensitive, and scalable technology that addresses the important area of RNA--protein interactions. Using aptamer-tagged RNA as bait, we captured RNA-interacting proteins from stable isotope labeling by amino acids in cell culture (SILAC)-labeled mammalian cell extracts and analyzed them by high-resolution, quantitative MS. Binders specific to the RNA sequence were distinguished from background by their isotope ratios between bait and control. We demonstrated the approach by retrieving known and novel interaction partners for the HuR interaction motif, H4 stem loop, 'zipcode' sequence, tRNA, and a bioinformatically-predicted RNA fold in DGCR-8/Pasha mRNA. In all experiments we unambiguously identified known interaction partners by a single affinity purification step. The 5' region of the mRNA of DGCR-8/Pasha, a component of the microprocessor complex, specifically interacts with components of the translational machinery, suggesting that it contains an internal ribosome entry site. quantitative mass spectrometry | ribonucleoprotein | RNA-binding proteins | HuR | internal ribosome entry site

Acute lung injury involving extremely immature lungs often heals without excessive fibrosis unlike later in gestation and in adults. Several factors may be involved, but fibroblast contraction of collagen has been linked to the level of wound fibrosis. To assess whether human lung fibroblasts of fetal versus adult origin differ in ability to contract collagen and define the molecular underpinnings, we performed three-dimensional collagen contraction assay, analyzed their differential mRNA profile, specifically for transforming growth factor-[beta] (TGF-[beta]) signaling pathway and extracellular matrix components, studied the cell response to TGF-[beta] in culture, and used two-dimensional gel electrophoresis followed by mass spectrometry to identify differences in their overall proteomes. Human lung fetal fibroblasts contracted the collagen matrix less than the adults. Smooth muscle actin expression did not differ. TGF-[beta] stimulation resulted in greater Smad3 phosphorylation in fetal compared with adults, mRNA and proteomic profiling reveal a number of TGF-[beta] pathways, ECM components, and cytoskeletal regulatory molecules are differentially expressed between the cell types. Of note is TGF-[beta] receptor interacting protein 1 (TRIP-l), which we show inhibits fibroblast collagen contraction and is higher in fetal than adult fibroblasts. We conclude that human lung fetal fibroblasts are less able to contract collagen than adult lung fibroblasts. The diminished ability is not due to impediment of Smad3 activation but rather, at least in part, due to their higher level of TRIP- 1 expression. TRIP-1 is a novel modulator of fibroblast collagen contraction. lung fibrosis; lung injury; lung repair; contractibility; proteomics; TGF-[beta] receptor interacting protein 1

Disease processes or exposure to environmental toxicants can produce tiny modifications (called adducts) on proteins in the blood. A clinical assay that reliably detects those modifications in plasma or serum [...]

Human tracheobronchial epithelial cells grown in air-liquid interface culture have emerged as a powerful tool for the study of airway biology. In this study, we have investigated whether this culture system produces ''mucus' with a protein composition similar to that of in vivo. induced airway secretions. Previous compositional studies of mucous secretions have greatly underrepresented the contribution of mucins, which are major structural components of normal mucus. To overcome this limitation, we have used a mass spectrometry-based approach centered on prior separation of the mucins from the majority of the other proteins. Using this approach, we have compared the protein composition of apical secretions (AS) from well-differentiated primary human tracheobronchial cells grown at air-liquid interface and human tracheobronchial normal induced sputum (IS). A total of 186 proteins were identified, 134 from AS and 136 from IS; 84 proteins were common to both secretions, with host defense proteins being predominant. The epithelial mucins MUC1, MUC4, and MUC16 and the gel-forming mucins MUC5B and MUC5AC were identified in both secretions. Refractometry showed that the gel-forming mucins were the major contributors by mass to both secretions. When the composition of the IS was corrected for proteins that were most likely derived from saliva, serum, and migratory cells, there was considerable similarity between the two secretions, in particular, in the category of host defense proteins, which includes the mucins. This shows that the primary cell culture system is an important model for study of aspects of innate defense of the upper airways related specifically to mucus consisting solely of airway cell products. mucus; mucin; innate immunity; proteomics; human tracheobronchial epithelial cell culture

Proteomics research is concerned with the analysis of all proteins found in an organism, tissue, cell type, or cellular structure. The shotgun proteomic approach, which involves two-dimensional gel electrophoresis or [...]

We have developed a multianalyte fluid-phase protein array technology termed high-throughput immunophenotyping using transcription (HIT). This method employs a panel of monoclonal antibodies, each tagged with a unique oligonucleotide sequence that serves as a molecular bar code. After staining a sample, T7 polymerase amplifies the tags, which are then hybridized to a DNA microarray for indirect measurement of each analyte. Although there are many potential applications for this technology, here we report its suitability for profiling cytokines, intracellular molecules and cell surface markers. Using HIT, we profiled 90 surface markers on human naive T helper cells activated in vitro. The markers identified in this screen are consistent with previously described activation markers and were validated by flow cytometry. Additionally, a HIT screen of surface markers expressed on T helper cells activated in the presence of transforming growth factor-[beta] identified downregulation of CD26 in these cells. HIT arrays are an ideal platform for rapidly identifying markers for further characterization and therapeutic intervention., Proteomics and genomics technologies offer tremendous promise for generating and testing new hypotheses related to disease and basic cell biology (1). In the field of proteomics, there is still a [...]

Steroid receptors belonging to the superfamily of nuclear receptors do not exist as single monomeric proteins but mediate their effects by the interaction with numerous other proteins, e.g., cofactors for transcription, but also other proteins involved in cellular signaling. This interaction may be ligand dependent, which explains the differential effects of receptor ligands. Whereas some receptors, e.g., the estrogen receptor, have been studied in great detail, much less is known about proteins interacting with the mineralocorticoid receptor (MR). In this study, we aimed to identify interacting proteins using a proteomics approach involving tagged receptor constructs. After affinity isolation of MR complexes, blue native electrophoresis revealed the presence of several populations of MR complexes differing in size and composition. During the identification of interacting proteins, various heat shock proteins but also several previously undescribed potential interactors were found, including 14-3-3-[epsilon]. We also demonstrate here that the cytosolic MR in the presence of detergent interacts in a ligand-selective manner with glucose-regulated protein 78 and propionyl-CoA carboxylase-[beta] precursor, which are found in the unliganded or aldosterone-containing complex but not with spironolactone. steroid receptor; aldosterone; mineralocorticoid; spironolactone; 143-3; proteomics

[beta]-arrestins are cytosolic proteins that form complexes with seven-transmembrane receptors after agonist stimulation and phosphorylation by the G protein-coupled receptor kinases. They play an essential role in receptor desensitization and endocytosis, and they also serve as receptor-regulated signaling scaffolds and adaptors. Moreover, in the past decade, a growing list of protein-protein interactions of [beta]-arrestins pertinent to these functions has been documented. The discovery of several novel functions of [beta]-arrestins stimulated us to perform a global proteomics analysis of [beta]-arrestin-interacting proteins (interactome) as modulated by a model seven-transmembrane receptor, the angiotensin II type la receptor, in an attempt to assess the full range of functions of these versatile molecules. As determined by LC tandem MS, 71 proteins interacted with [beta]-arrestin 1, 164 interacted with [beta]-arrestin 2, and 102 interacted with both [beta]-arrestins. Some proteins bound only after agonist stimulation, whereas others dissociated. Bioinformatics analysis of the data indicates that proteins involved in cellular signaling, organization, and nucleic acid binding are the most highly represented in the [beta]-arrestin interactome. Surprisingly, both S-arrestin (visual arrestin) and X-arrestin (cone arrestin) were also found in heteromeric complex with [beta]-arrestins. The [beta]-arrestin interactors distribute not only in the cytoplasm, but also in the nucleus as well as other subcellular compartments. The binding of 16 randomly selected newly identified [beta]-arrestin partners was validated by coimmunoprecipitation assays in HEK293 cells. This study provides a comprehensive analysis of proteins that bind [beta]-arrestin isoforms and underscores their potentially broad regulatory roles in mammalian cellular physiology. mass spectrometry | seven-transmembrane receptor | angiotensin II type la receptor | interactome | signal transduction

Myxococcus xanthus is a soil-dwelling, gram-negative bacterium that during nutrient deprivation is capable of undergoing morphogenesis from a vegetative rod to a spherical, stress-resistant spore inside a domed-shaped, multicellular fruiting body. To identify proteins required for building stress-resistant M. xanthus spores, we compared the proteome of liquid-grown vegetative cells with the proteome of mature fruiting body spores. Two proteins, protein S and protein S1, were differentially expressed in spores, as has been reported previously. In addition, we identified three previously uncharacterized proteins that are differentially expressed in spores and that exhibit no homology to known proteins. The genes encoding these three novel major spore proteins (mspA, mspB, and mspC) were inactivated by insertion mutagenesis, and the development of the resulting mutant strains was characterized. All three mutants were capable of aggregating, but for two of the strains the resulting fruiting bodies remained flattened mounds of cells. The most pronounced structural defect of spores produced by all three mutants was an altered cortex layer. We found that mspA and mspB mutant spores were more sensitive specifically to heat and sodium dodecyl sulfate than wild-type spores, while mspC mutant spores were more sensitive to all stress treatments examined. Hence, the products of mspA, mspB, and mspC play significant roles in morphogenesis of M. xanthus spores and in the ability of spores to survive environmental stress.

Osmoregulated periplasmic glucans (OPGs) are general constituents of the envelope of Gram-negative bacteria. They are required for full virulence of bacterial phytopathogens such as Pseudomonas syringae, Xanthomonas campestris and Erwinia chrysanthemi. E. chrysanthemi is a pectinolytic [gamma]-proteobacterium that causes soft rot disease on a wide range of plant species. In addition to the loss of virulence, opg mutants exhibit a pleiotropic phenotype that affects motility, bile-salt resistance, exoenzyme secretion, exopolysaccharide synthesis and membrane lipid composition. This is believed to be the first proteomic analysis of an OPG-defective mutant of E. chrysanthemi and it revealed that, in addition to the effects described, catabolic enzyme synthesis was enhanced and there was a greater abundance of some proteins catalysing the folding and degradation of proteins needed for various stress responses. Thus, in the opg mutant strain, loss of virulence was the result of a combination of envelope structure changes and cellular metabolism modifications.

Components of multiprotein complexes are routinely determined by using proteomic approaches. However, this information lacks functional content except when new complex members are identified. To analyze quantitatively the abundance of proteins in human Mediator we used normalized spectral abundance factors generated from shotgun proteomics data sets. With this approach we define a common core of mammalian Mediator subunits shared by alternative forms that variably associate with the kinase module and RNA polymerase (pol) II. Although each version of affinity-purified Mediator contained some kinase module and RNA pol II, Mediator purified through F-Med26 contained the most RNA pol II and the least kinase module as demonstrated by the normalized spectral abundance factor approach. The distinct forms of Mediator were functionally characterized by using a transcriptional activity assay, where F-Med26 Mediator/RNA pol II was the most active. This method of protein complex visualization has important implications for the analysis of multiprotein complexes and assembly of protein interaction networks. multidimensional protein identification technology | proteomics | spectrum counting | mass spectrometry

There are ~100,000 proteins in humans with various physiological functions. The complement of proteins in the organism as well as their interactions is defined as the proteome. Its analysis (proteomics) by highly specific, sensitive, and accurate MS has been made possible with matrix-assisted laser desorption ionization or electrospray ionization of proteins and large peptides. Currently, the most commonly used proteomics technologies involve either specific digestion of proteins (the bottom-up approach using 2-dimensional polyacrylamide gel electrophoresis and multidimensional protein identification technology) or direct analysis of intact proteins after their chromatographic separation (the top-down approach and surface-enhanced laser desorption ionization). Proteomics holds great promise for discoveries in nutrition research, including profiles and characteristics of dietary and body proteins; digestion, absorption, and metabolism of nutrients; functions of nutrients and other dietary factors in growth, reproduction, and health; biomarkers of the nutritional status and disease; and individualized requirements of nutrients. The proteome analysis is expected to play an important role in solving major nutrition-associated problems in humans and animals, such as obesity, diabetes, cardiovascular disease, cancer, aging, and intrauterine fetal retardation. KEY WORDS: * proteome * nutrients * metabolism * growth * health * disease

Abstract: The term proteomics, although still less than a decade old, is becoming commonplace in the vocabulary of biologists. Advances made in yeast and humans have been remarkable, sustained by [...]

Epidemiologic and experimental studies showed that folate deficiency is associated with increased risk of degenerative diseases by enhancing abnormal one-carbon metabolism. We studied the changes in the proteome of liver, the main tissue of folate storage and metabolism, in a rat model of dietary folate depletion. Four-month-old rats were fed for 4 wk an amino acid-defined diet without folate and compared with pair-fed rats given the same diet adequately supplemented with folic acid. Folate deprivation decreased plasma and hepatic folate concentrations dramatically, while increasing homocysteinemia significantly. Using 2-dimensional electrophoresis and matrix-assisted laser desorption/ionization time-of-flight MS, we identified 9 spots corresponding to differentially expressed proteins in the liver of folate-deficient rats compared with controls. Among those spots, 4 had a significantly increased volume, whereas the volume of the 5 other spots was decreased. Upregulated proteins included glutathione peroxidase (GPx) 1 and peroxiredoxin 6, 2 enzymes involved in the response to oxidative stress, and MAWD binding protein (MAWDBP), which has been associated with cancer. MAWDBP was simultaneously identified as a second spot with a lower isoelectric point (p/) that vanished almost completely after folate deficiency. Decreased abundance was also observed for cofilin 1, a protein linked to tumorigenesis, and for the GRP 75 precursor and preproalbumin, both of which are responsive to oxidative stress and/or inflammation. Moreover, an enzyme activity assay and/or Western blot analysis of GPx-1 and MAWDBP confirmed the proteomic findings. Our results show that folate deficiency modifies the abundance of several liver proteins consistently with adaptive tissue responses to oxidative and degenerative processes. KEY WORDS: * proteomics * antioxidant enzymes * homocysteine * hepatic proteins * folate deprivation

As the sole site of nucleocytoplasmic transport, the nuclear pore complex (NPC) has a vital cellular role. Nonetheless, much remains to be learned about many fundamental aspects of NPC function. To further understand the structure and function of the mammalian NPC, we have completed a proteomic analysis to identify and classify all of its protein components. We used mass spectrometry to identify all proteins present in a biochemically purified NPC fraction. Based on previous characterization, sequence homology, and subcellular localization, 29 of these proteins were classified as nucleoporins, and a further 18 were classified as NPC-associated proteins. Among the 29 nucleoporins were six previously undiscovered nucleoporins and a novel family of WD repeat nucleoporins. One of these WD repeat nucleoporins is ALADIN, the gene mutated in triple-A (or Allgrove) syndrome. Our analysis defines the proteome of the mammalian NPC for the first time and paves the way for a more detailed characterization of NPC structure and function.

In this study, a proteomic approach was applied for the generation of reference maps and subsequently to detect, quantify and compare the global protein expression between two related species of marine mussels, Mytilus edulis and Mytilus galloprovincialis, growing in their own geographical habitats. A comparative study of the protein profiles generated from analytical two-dimensional electrophoresis gels was performed, and changes in protein expression were analysed quantitatively by computer analysis. An average of 1,278 spots per gel was detected in 16 individuals (8 M. edulis and 8 M. galloprovincialis); however, not all spots were included in the study. Expression of 420 spots was compared, and significant differences in the intensity levels were detected in 37 protein spots (8.8%). Fifteen proteins showed higher expression in M. edulis, and 22 proteins, in M. galloprovincialis. The technique of peptide mass fingerprinting using MALDI-TOF (matrix-assisted laser desorption ionisation/time-of-flight) and/ or nanoelectrospray double subfragmentation mass spectrometry enabled the unambiguous identification of 15 of these 37 differentially expressed proteins. Most of the identified proteins can be grouped basically into four broad functional classes: cytoskeletal and myofibrillar proteins, proteins associated with stress response, proteins associated with the storage or production of energy, and proteins related to rearrangement in the synthesis of native structures. These results expand our understanding of the molecular differentiation of the two mussel taxa and serve as a useful base for future ecological, physiological and genetic studies.