Deamidation at Asparagine and Glutamine As a Major Modification upon Deterioration/Aging of Proteinaceous Binders in MuralPaintings

Proteomic strategies are herein proved to be a complementary approach to the well established amino acid composition analysis for the characterization of the aging and deterioration phenomena occurring to proteinaceous materials in works-of-art. Amino acid analyses on several samples demonstrated that proteins in the frescoes from the Camposanto Monumentale in Pisa are deteriorated as revealed by the decrease in Met, Lys, and Tyr content and by the presence in all the samples of amino malonic acid as a result of Ser, Phe, and Cys oxidation. Proteomic analysis identified deamidation at Asn and Gln as a further major event occurred. This work paves the way to the exploitation of proteomic strategies for the investigation of the molecular effects of aging and deterioration in historical objects. Results show that proteomic searches for deamidation by liquid chromatography-tandem mass spectrometry (LC-MS/MS) could constitute a routine analysis for paintings or any artistic and historic objects where proteins are present. Peptides that can be used as molecular markers when casein is present were identified

Broad spectrum proteomics analysis of the inferior colliculus following acute hydrogen sulfide exposure.

Acute exposure to high concentrations of H2S causes severe brain injury and long-term neurological disorders, but the mechanisms involved are not known. To better understand the cellular and molecular mechanisms involved in acute H2S-induced neurodegeneration we used a broad-spectrum proteomic analysis approach to identify key molecules and molecular pathways involved in the pathogenesis of acute H2S-induced neurotoxicity and neurodegeneration. Mice were subjected to acute inhalation exposure of up to750 ppm of H2S. H2S induced behavioral deficits and severe lesions including hemorrhage in the inferior colliculus (IC). The IC was microdissected for proteomic analysis. Tandem mass tags (TMT) liquid chromatography mass spectrometry (LC-MS/MS)-based quantitative proteomics was applied for protein identification and quantitation. LC-MS/MS identified 598, 562, and 546 altered proteomic changes at 2 h, and on days 2 and 4 post-H2S exposure, respectively. Of these, 77 proteomic changes were statistically significant at any of the 3 time points. Mass spectrometry data were subjected to Perseus 1.5.5.3 statistical analysis, and gene ontology heat map clustering. Expressions of several key molecules were verified to confirm H2S-dependent proteomics changes. Webgestalt pathway overrepresentation enrichment analysis with Panther engine revealed H2S exposure disrupted several biological processes including metabotropic glutamate receptor group 1 and inflammation mediated by chemokine and cytokine signaling pathways among others. Further analysis showed that energy metabolism, integrity of blood-brain barrier, hypoxic, and oxidative stress signaling pathways were also implicated. Collectively, this broad-spectrum proteomics data has provided important clues to follow up in future studies to further elucidate mechanisms of H2S-induced neurotoxicity

SILAC-based proteomic quantification of chemoattractant-induced cytoskeleton dynamics on a second to minute timescale

Cytoskeletal dynamics during cell behaviours ranging from endocytosis and exocytosis to cell division and movement is controlled by a complex network of signalling pathways, the full details of which are as yet unresolved. Here we show that SILAC-based proteomic methods can be used to characterize the rapid chemoattractant-induced dynamic changes in the actin–myosin cytoskeleton and regulatory elements on a proteome-wide scale with a second to minute timescale resolution. This approach provides novel insights in the ensemble kinetics of key cytoskeletal constituents and association of known and novel identified binding proteins. We validate the proteomic data by detailed microscopy-based analysis of in vivo translocation dynamics for key signalling factors. This rapid large-scale proteomic approach may be applied to other situations where highly dynamic changes in complex cellular compartments are expected to play a key role

Joint single-cell DNA accessibility and protein epitope profiling reveals environmental regulation of epigenomic heterogeneity.

Here we introduce Protein-indexed Assay of Transposase Accessible Chromatin with sequencing (Pi-ATAC) that combines single-cell chromatin and proteomic profiling. In conjunction with DNA transposition, the levels of multiple cell surface or intracellular protein epitopes are recorded by index flow cytometry and positions in arrayed microwells, and then subject to molecular barcoding for subsequent pooled analysis. Pi-ATAC simultaneously identifies the epigenomic and proteomic heterogeneity in individual cells. Pi-ATAC reveals a casual link between transcription factor abundance and DNA motif access, and deconvolute cell types and states in the tumor microenvironment in vivo. We identify a dominant role for hypoxia, marked by HIF1α protein, in the tumor microvenvironment for shaping the regulome in a subset of epithelial tumor cells

Proteomic analysis of the bovine and human ciliary zonule

PURPOSE: The zonule of Zinn (ciliary zonule) is a system of fibers that centers the crystalline lens on the optical axis of the eye. Mutations in zonule components underlie syndromic conditions associated with a broad range of ocular pathologies, including microspherophakia and ectopia lentis. Here, we used HPLC–mass spectrometry to determine the molecular composition of the zonule. METHODS: Tryptic digests of human and bovine zonular samples were analyzed by HPLC–mass spectrometry. The distribution of selected components was confirmed by immunofluorescence confocal microscopy. In bovine samples, the composition of the equatorial zonule was compared to that of the hyaloid zonule and vitreous humor. RESULTS: The 52 proteins common to the zonules of both species accounted for >95% of the zonular protein. Glycoproteins constituted the main structural components, with two proteins, FBN1 and LTBP2, constituting 70%–80% of the protein. Other abundant components were MFAP2, EMILIN-1, and ADAMTSL-6. Lysyl oxidase-like 1, a crosslinking enzyme implicated in collagen and elastin biogenesis, was detected at significant levels. The equatorial and hyaloid zonular samples were compositionally similar to each other, although the hyaloid sample was relatively enriched in the proteoglycan opticin and the fibrillar collagens COL2A1, COL11A1, COL5A2, and COL5A3. CONCLUSIONS: The zonular proteome was surprisingly complex. In addition to structural components, it contained signaling proteins, protease inhibitors, and crosslinking enzymes. The equatorial and hyaloid zonules were similar in composition, but the latter may form part of a composite structure, the hyaloid membrane, that stabilizes the vitreous face

Proteomic analysis of skin invasion by blood fluke larvae.

BackgroundDuring invasion of human skin by schistosome blood fluke larvae (cercariae), a multicellular organism breaches the epidermis, basement membrane, and dermal barriers of skin. To better understand the pathobiology of this initial event in schistosome infection, a proteome analysis of human skin was carried out following invasion by cercariae of Schistosoma mansoni.Methodology and resultsHuman skin samples were exposed to cercariae for one-half hour to two hours. Controls were exposed to water used to collect cercariae in an identical manner, and punctured to simulate cercarial tunnels. Fluid from both control and experimental samples was analyzed by LC/MS/MS using a linear ion trap in "triple play" mode. The coexistence of proteins released by cercariae and host skin proteins from epidermis and basement membrane confirmed that cercarial tunnels in skin were sampled. Among the abundant proteins secreted by cercariae was the cercarial protease that has been implicated in degradation of host proteins, secreted proteins proposed to mediate immune invasion by larvae, and proteins implicated in protection of parasites against oxidative stress. Components of the schistosome surface tegument, previously identified with immune serum, were also released. Both lysis and apoptosis of epidermal cells took place during cercarial invasion of the epidermis. Components of lysed epidermal cells, including desmosome proteins which link cells in the stratum granulosum and stratum spinosum, were identified. While macrophage-derived proteins were present, no mast cell or lymphocyte cytokines were identified. There were, however, abundant immunoglobulins, complement factors, and serine protease inhibitors in skin. Control skin samples incubated with water for the same period as experimental samples ensured that invasion-related proteins and host protein fragments were not due to nonspecific degeneration of the skin samples.ConclusionsThis analysis identified secreted proteins from invasive larvae that are released during invasion of human skin. Analysis of specific host proteins in skin invaded by cercariae served to highlight both the histolytic events facilitating cercarial invasion, and the host defenses that attempt to arrest or retard invasion. Proteins abundant in psoriatic skin or UV and heat-stressed skin were not abundant in skin invaded by cercariae, suggesting that results did not reflect general stress in the surgically removed skin specimen. Abundant immunoglobulins, complement factors, and serine protease inhibitors in skin form a biochemical barrier that complements the structural barrier of the epidermis, basement membrane, and dermis. The fragmentation of some of these host proteins suggests that breaching of host defenses by cercariae includes specific degradation of immunoglobulins and complement, and either degradation of, or overwhelming the host protease inhibitor repertoire

Rapid pre-gel visualization of proteins with mass spectrometry compatibility

Despite all of the prophecies of doom, gel electrophoresis is still prevalent in modern proteomic workflows. However, the currently used protein staining methods represent a serious bottleneck for a quick subsequent protein analysis using mass spectrometry. Substituting traditional protein stains by pre-gel derivatization with visible and mass spectrometry compatible reagents eliminates several processing steps and drastically reduces the sample preparation time. A defined chemistry permits seamless integration of such covalent protein staining methods into standardized bioinformatic pipelines. Using Uniblue A we could covalently stain simple to complex protein samples within 1 minute. Protein profiles on the gels were not compromised and MS/MS based sequence coverages higher than 80% could be obtained. In addition, the visual tracking of covalently stained proteins and peptides facilitates method development and validation. Altogether, this new chemo-proteomic approach enables true "at-line" analysis of proteins

PROTEOMIC ANALYSIS OF TWO DIFFERENT STATES OF NAEGLERIA FOWLERI

Naegleria fowleri are free-living ameboflagellates found in soil and freshwater habitats throughout the world that cause a fatal disease in humans called Primary Amoebic Meningoencephalitis (PAM). Mechanisms of host resistance or susceptibility to infection have not been fully elucidated, and possible treatment methods are still sub optimal. The disease is diagnosed using specific laboratory tests available in only a few laboratories in the United States. Because of the rarity of infection and difficulty in initial detection, more than often PAM is misdiagnosed. Therefore, it is very important to find causative marker for early detection of an infection. The purpose of this study is to create a proteomic signature map using two-dimensional gel electrophoresis (2-D gel) and recommend a subset of proteins that may be directly linked to the pathogenic state of N. fowleri. Replicates of 2-D Gels were created for both strains of N. fowleri and the proteomic templates from these gels were compared with each other. Scatter Plots were generated measuring the density of protein spots from 2-D gels being analyzed for each study. For each strains of N. fowleri, the 2-D gels from each study were compared within and compared between the two studies for reproducibility in data. The resulting correlation values for all of the Scatter Plots were greater or equal to 0.90. Finally, the representative proteomic template for axenically grown N. fowleri and mouse passaged N. fowleri were compared and the correlation value of 0.60 was observed. This confirmed our theory that these two strains or states of N. fowleri have very different protein expressions, and we were able to identify a subset of proteins, both over expressed and newly synthesized, that may be linked to the highly pathogenic state of N. fowleri

Characteristics of the tomato chromoplast revealed by proteomic analysis

Chromoplasts are non-photosynthetic specialized plastids that are important in ripening tomato fruit (Solanum lycopersicum) since, among other functions, they are the site of accumulation of coloured compounds. Analysis of the proteome of red fruit chromoplasts revealed the presence of 988 proteins corresponding to 802 Arabidopsis unigenes, among which 209 had not been listed so far in plastidial databanks. These data revealed several features of the chromoplast. Proteins of lipid metabolism and trafficking were well represented, including all the proteins of the lipoxygenase pathway required for the synthesis of lipid-derived aroma volatiles. Proteins involved in starch synthesis co-existed with several starch-degrading proteins and starch excess proteins. Chromoplasts lacked proteins of the chlorophyll biosynthesis branch and contained proteins involved in chlorophyll degradation. None of the proteins involved in the thylakoid transport machinery were discovered. Surprisingly, chromoplasts contain the entire set of Calvin cycle proteins including Rubisco, as well as the oxidative pentose phosphate pathway (OxPPP). The present proteomic analysis, combined with available physiological data, provides new insights into the metabolic characteristics of the tomato chromoplast and enriches our knowledge of non-photosynthetic plastids