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PubMed2005

Structural proteomics of macromolecular assemblies using oxidative footprinting and mass spectrometry.

Understanding the composition, structure and dynamics of macromolecules and their assemblies is at the forefront of biological science today. Hydroxyl-radical-mediated protein footprinting using mass spectrometry can define macromolecular structure, macromolecular assembly and conformational changes of macromolecules in solution based on measurements of reactivity of amino acid side-chain groups with covalent-modification reagents. Subsequent to oxidation by reactive oxygen species, proteins are digested by specific proteases to generate peptides for analysis by mass spectrometry. Accurate measurements of side-chain reactivity are achieved using quantitative liquid-chromatography-coupled mass spectrometry, whereas the side-chain sites within the macromolecular probes are identified using tandem mass spectrometry. In addition, the use of footprinting data in conjunction with computational modeling approaches is a powerful new method for testing and refining structural models of macromolecules and their complexes.

Chromatography, Liquid↗
PubMed2006

Merger of laser capture microdissection and mass spectrometry: a window into the amyloid plaque proteome.

The occurrence of protein accumulation and aggregation in the brain is one of the pathological hallmarks of neurodegenerative diseases such as Alzheimer's disease (AD). Although it is instructive to analyze the aggregated proteins in the brain, biochemical purification and identification of these proteins have been challenging. Recent developments in laser capture microdissection (LCM) and mass spectrometry (MS) enable large-scale protein profiling of captured tissue samples. We present here the method of analyzing senile plaques from postmortem AD brains by coupling LCM and highly sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS). First, the senile plaques were stained with thioflavin-S and precisely isolated by adjusted laser beams under a microscope. Total proteins in the isolated tissues were extracted and resolved on an SDS gel. To identify all proteins in the samples, the gel was excised into multiple pieces followed by trypsin digestion. The resulting peptides were further separated by reverse-phase chromatography and analyzed by tandem mass spectrometry. A database search of acquired MS/MS spectra allowed the identification of hundreds to thousands of peptides/proteins in the original samples. Moreover, quantitative comparison of protein composites in different LCM samples could be achieved by MS strategies. For instance, the comparison between plaques and surrounding nonplaque tissues from the same specimen revealed tens of proteins specifically enriched in the plaques. Finally, the data were corroborated by independent experiments using the approach of immunohistochemistry. Taken together, the merger of LCM and MS is a powerful tool to probe the proteome of any given pathological lesions.

Animals↗
PubMed2002

The proteomics of senescence in leaves of white clover, Trifolium repens (L.).

A proteomics approach has been used to study changes in protein abundance during leaf senescence in white clover. Changes in cell ultrastructure were also examined using transmission electron microscopy. The most obvious ultrastructural changes during senescence occurred in chloroplasts, with progressive loss of thylakoid integrity and accumulation of osmiophilic globules in the stroma. Quantitative analysis of 590 leaf protein spots separated by two-dimensional electrophoresis indicated that approximately 40% of the spots showed significant senescence related changes in abundance. Approximately one-third of the protein spots present in mature green leaves were also visible by two-dimensional electrophoresis of an isolated chloroplast fraction, and these spots represented a major proportion of the proteins showing senescence related declines in abundance. Chloroplast proteins that were identified by matrix-assisted laser desorption/ionization-time of flight mass fingerprinting included rubisco large and small subunits, a rubisco activase and the 33 kDa protein of the photosystem II oxygen-evolving complex. These proteins declined in abundance late in senescence, indicating that the photosynthetic apparatus was being degraded. A chloroplast glutamine synthetase showed partial decline in abundance during late senescence but was maintained at levels that may support provision of glutamine for export to other tissues. The results emphasise the importance of proteolysis, chloroplast degradation and remobilisation of nitrogen in leaf senescence.

Cellular Senescence↗
PubMed2003

Enhanced resolution of glycosylphosphatidylinositol-anchored and transmembrane proteins from the lipid-rich myelin membrane by two-dimensional gel electrophoresis.

Two-dimensional gel electrophoresis (2-DE) has become a powerful and widely used technique for proteomic analyses. However, the limited ability of 2-DE to resolve transmembrane and glycosylphosphatidylinositol (GPI)-anchored proteins has slowed the identification of proteins from membrane-rich biological samples. Myelin is an unusually lipid-rich membrane with relatively few major proteins but many quantitatively minor proteins, most of which have an unknown identity and/or function. The goal of this study was to identify the optimal conditions of 2-DE for the separation of myelin proteins. We have identified two detergents, the nonionic n-dodecyl beta-D-maltoside and the zwitterionic amidosulfobetaine ASB-14, that are more effective in solubilizing myelin proteins than the commonly used zwitterionic detergent 3-[(3-cholamidopropyl)- dimethylammonio]-1-propanesulfonate (CHAPS). These detergents significantly enhance the solubility of both transmembrane (e.g., the highly hydrophobic and multiply acylated myelin proteolipid protein) and GPI-anchored (e.g., contactin and neuronal cell adhesion molecule) myelin proteins and enable their resolution by 2-DE. We conclude that these detergents are effective tools for the 2-DE analysis of myelin, and that they may be more generally useful for the analysis of membrane-rich biological samples.

Acetone↗
PubMed2004

Two-dimensional reference map of Agrobacterium tumefaciens proteins.

Proteomics based on two-dimensional (2-D) gel electrophoresis of proteins followed by spot identification with mass spectrometry is a commonly used method for physiological studies. Physiological proteomics requires 2-D reference maps, on which most of the main proteins are identified. We present a reference map for the bacterial plant pathogen Agrobacterium tumefaciens proteins, which contains more than 300 entries with an isoelectric point (pI) between 4 and 7. The quantitative study of the proteins in the analytical window of the master gel demonstrated unique features, in comparison with other bacteria. In addition, a theoretical analysis of several protein parameters was performed and compared with the experimental results. A comparison of the theoretical molecular weight (MW) of the proteins and their theoretical pI with their vertical and horizontal migration distances, respectively, pointed out the existence of several proteins that strongly diverted from the graph trend-line. These proteins were clearly subjected to post-translational modifications, which changed their pI and/or MW. Additional support for post-translational modifications comes from the identification of multiple spots of the same gene products. Post-translational modifications appear to be more common than expected, at least for soluble proteins, as more than 10% of the proteins were associated with multiple spots.

Agrobacterium tumefaciens↗
PubMed2024

Comprehensive Assessment of the Intrinsic Pancreatic Microbiome.

OBJECTIVE: To sought comprehensively profile tissue and cyst fluid in patients with benign, precancerous, and cancerous conditions of the pancreas to characterize the intrinsic pancreatic microbiome. BACKGROUND: Small studies in pancreatic ductal adenocarcinoma (PDAC) and intraductal papillary mucinous neoplasm (IPMN) have suggested that intrapancreatic microbial dysbiosis may drive malignant transformation. METHODS: Pancreatic samples were collected at the time of resection from 109 patients. Samples included tumor tissue (control, n = 20; IPMN, n = 20; PDAC, n = 19) and pancreatic cyst fluid (IPMN, n = 30; serous cystadenomas, n = 10; mucinous cystic neoplasm, n = 10). Assessment of bacterial DNA by quantitative polymerase chain reaction and 16S ribosomal RNA gene sequencing was performed. Downstream analyses determined the relative abundances of individual taxa between groups and compared intergroup diversity. Whole-genome sequencing data from 140 patients with PDAC in the National Cancer Institute's Clinical Proteomic Tumor Analysis Consortium were analyzed to validate findings. RESULTS: Sequencing of pancreatic tissue yielded few microbial reads regardless of diagnosis, and analysis of pancreatic tissue showed no difference in the abundance and composition of bacterial taxa between normal pancreas, IPMN, or PDAC groups. Low-grade and high-grade dysplasia IPMN were characterized by low bacterial abundances with no difference in tissue composition and a slight increase in Pseudomonas and Sediminibacterium in high-grade dysplasia cyst fluid. Decontamination analysis using the Clinical Proteomic Tumor Analysis Consortium database confirmed a low-biomass, low-diversity intrinsic pancreatic microbiome that did not differ by pathology. CONCLUSIONS: Our analysis of the pancreatic microbiome demonstrated very low intrinsic biomass that is relatively conserved across diverse neoplastic conditions and thus unlikely to drive malignant transformation.

Humans↗
PubMed2006

Increases in pig major acute-phase protein in wasting pigs brought to the abattoir.

Plasma proteins of wasting pigs were quantitatively and qualitatively compared with those of normal fattening pigs. Higher expression of a 120 kDa protein was observed in the plasma of wasting pigs by SDS-PAGE. This protein was identified as pig major acute-phase protein (Pig-MAP) by proteomic analysis using two-dimensional electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The plasma concentration of Pig-MAP in wasting pigs was 7-fold higher than that of normal ones.

Abattoirs↗
PubMedDate not supplied

Intact-protein based sample preparation strategies for proteome analysis in combination with mass spectrometry.

The complexity of tissue and cell proteomes and the vast dynamic range of protein abundance present a formidable challenge for analysis that no one analytical technique can overcome. As a result, there is a need to integrate technologies to achieve the high-resolution and high-sensitivity analysis of complex biological samples. The combined technologies of separation science and biological mass spectrometry (Bio-MS) are the current workhorse in proteomics, and are continuing to evolve to meet the needs for high sensitivity and high throughput. They are relied upon for protein quantification, identification, and analysis of post-translational modifications (PTMs). The standard technique of two dimensional poly-acrylamide gel electrophoresis (2D PAGE) offers relatively limited resolution and sensitivity for the simultaneous analysis of all cellular proteins, with only the most highly abundant proteins detectable in whole cell or tissue-derived samples. Hence, many alternative strategies are being explored. Numerous sample preparation procedures are currently available to reduce sample complexity and to increase the detectability of low-abundance proteins. Maintaining proteins intact during sample preparation has important advantages compared with strategies that digest proteins at an early step. These strategies include the ability to quantitate and recover proteins, and the assessment of PTMs. A review of current intact protein-based strategies for protein sample preparation prior to mass spectrometry (MS) is presented in the context of biomedically driven applications.

Animals↗
PubMed2005

How molecular profiling could revolutionize drug discovery.

Information from genomic, proteomic and metabolomic measurements has already benefited target discovery and validation, assessment of efficacy and toxicity of compounds, identification of disease subgroups and the prediction of responses of individual patients. Greater benefits can be expected from the application of these technologies on a significantly larger scale; by simultaneously collecting diverse measurements from the same subjects or cell cultures; by exploiting the steadily improving quantitative accuracy of the technologies; and by interpreting the emerging data in the context of underlying biological models of increasing sophistication. The benefits of applying molecular profiling to drug discovery and development will include much lower failure rates at all stages of the drug development pipeline, faster progression from discovery through to clinical trials and more successful therapies for patient subgroups. Upheavals in existing organizational structures in the current 'conveyor belt' models of drug discovery might be required to take full advantage of these methods.

Animals↗
PubMed2002

Two antigenic peptides from genes m123 and m164 of murine cytomegalovirus quantitatively dominate CD8 T-cell memory in the H-2d haplotype.

The importance of CD8 T cells for the control of cytomegalovirus (CMV) infection has raised interest in the identification of immunogenic viral proteins as candidates for vaccination and cytoimmunotherapy. The final aim is to determine the viral "immunome" for any major histocompatibility complex class I molecule by antigenicity screening of proteome-derived peptides. For human CMV, there is a limitation to this approach: the T cells used as responder cells for peptide screening are usually memory cells that have undergone in vivo selection. On this basis, pUL83 (pp65) and pUL123 (IE1 or pp68 to -72) were classified as immunodominant proteins. It is an open question whether this limited "memory immunome" really reflects the immunogenic potential of the human CMV proteome. Here we document an analogous focus of the memory repertoire on two proteins of murine CMV. Specifically, ca. 80% of all memory CD8 T cells in the spleen as well as in persisting pulmonary infiltrates were found to be specific for the known IE1 peptide 168YPHFMPTNL176 and for the peptide 257AGPPRYSRI265, newly defined here, derived from open reading frame m164. Notably, CD8 T-cell lines of both specificities protected against acute infection upon adoptive transfer. In contrast, the natural immune response to acute infection in draining lymph nodes and in the lungs indicated a somewhat broader specificity repertoire. We conclude that the low number of antigenic peptides identified so far for CMVs reflects a focused memory repertoire, and we predict that more antigenic peptides will be disclosed by analysis of the acute immune response.

Animals↗
PubMedDate not supplied

Utilizing human blood plasma for proteomic biomarker discovery.

Candidate proteomic biomarker discovery from human plasma holds both incredible clinical potential as well as significant challenges. The dynamic range of proteins within plasma is known to exceed 10(10), and many potential biomarkers are likely present at lower protein abundances. At present, proteomic based MS analyses provide a dynamic range typically not exceeding approximately 10(3) in a single spectrum, and approximately 10(4)-10(6) when combined with on-line separations (e.g., reversed-phase gradient liquid chromatography), and thus are generally insufficient for low level biomarker detection directly from human plasma. This limitation is providing an impetus for the development of experimental methodologies and strategies to increase the possible number of detections within this biofluid. Discussed is the diversity of available approaches currently used by our laboratory and others to utilize human plasma as a viable medium for biomarker discovery. Various separation, depletion, enrichment, and quantitative efforts as well as recent improvements in MS capabilities have resulted in measurable improvements in the detection and identification of lower abundance proteins (by approximately 10-10(2)). Despite these improvements, further advances are needed to provide a basis for discovery of candidate biomarkers at very low levels. Continued development of depletion and enrichment techniques, coupled with improved pre-MS separations (both at the protein and peptide level) holds promise in extending the dynamic range of proteomic analysis.

Biomarkers↗
PubMed2006

Database of two-dimensional polyacrylamide gel electrophoresis of proteins labeled with CyDye DIGE Fluor saturation dye.

CyDye DIGE Fluor saturation dye (saturation dye, GE Healthcare Amersham Biosciences) enables highly sensitive 2-D PAGE. As the dye reacts with all reduced cysteine thiols, 2-D PAGE can be performed with a lower amount of protein, compared with CyDye DIGE Fluor minimal dye (GE Healthcare Amersham Biosciences), the sensitivity of which is equivalent to that of silver staining. We constructed a 2-D map of the saturation dye-labeled proteins of a liver cancer cell line (HepG2) and identified by MS 92 proteins corresponding to 123 protein spots. Functional classification revealed that the identified proteins had chaperone, protein binding, nucleotide binding, metal ion binding, isomerase activity, and motor activity. The functional distribution and the cysteine contents of the proteins were similar to those in the most comprehensive 2-D database of hepatoma cells (Seow et al.., Electrophoresis 2000, 21, 1787-1813), where silver staining was used for protein visualization. Hierarchical clustering on the basis of the quantitative expression profiles of the 123 characterized spots labeled with two charge- and mass-matched saturation dyes (Cy3 and Cy5) discriminated between nine hepatocellular carcinoma cell lines and primary cultured hepatocytes from five individuals, suggesting the utility of saturation dye and our database for proteomic studies of liver cancer.

Cell Line, Tumor↗
PubMed2026

Dictamnine alleviates oxidative stress in rheumatoid arthritis via modulation of the NR1D1-Keap1/Nrf2/ARE axis.

Rheumatoid arthritis (RA) is a persistent systemic disorder of autoimmune origin, with its core pathological manifestation being inflammation of the synovial tissue. The excessive growth of fibroblast-like synoviocytes (FLS) represents a critical pathological mechanism in RA, actively driving the advancement of the condition. Dictamnus dasycarpus Turcz. (D. dasycarpus) exhibits prominent anti-inflammatory effects and shows favorable therapeutic efficacy against RA. Dictamnine (Dic) is a major active component of D. dasycarpus, however, its therapeutic effectiveness and underlying mechanisms in RA have yet to be fully elucidated. This study investigated the effect of Dic on synovial hyperplasia in RA and elucidated the underlying mechanisms. Using a TNF-α-induced human fibroblast-like synoviocyte (HFLS-RA) model and a collagen-induced arthritis (CIA) mouse model, Dic was found to effectively inhibit synovial cell proliferation and pathological hyperplasia. Proteomics analysis was employed to clarify its potential mechanism in ameliorating the disease, and the findings were further validated through hematoxylin and eosin (H&E) staining, immunofluorescence (IF), ROS detection, JC-1 staining, cellular thermal shift assay (CETSA), drug affinity responsive target stability (DARTS) analysis, quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting (WB). The results suggested that the anti-RA activity of Dic is associated with its interaction with the nuclear receptor NR1D1. Moreover, the NR1D1 antagonist SR8278 reversed Dic's effects on Nrf2 and cytoprotection, confirming that Dic functions through NR1D1. This activation consequently influences the Keap1/Nrf2/ARE cascade, leading to decreased intracellular reactive oxygen species (ROS) accumulation and an improvement in compromised mitochondrial membrane potential. siRNA knockdown experiments further confirmed that NR1D1 is a target of Dic and regulates the downstream Keap1/Nrf2/HO-1 signaling pathway, through which Dic ameliorates RA both in vitro and in vivo by upregulating NR1D1 expression to activate the Keap1/Nrf2/ARE antioxidant pathway, thereby mitigating oxidative stress, inhibiting synovial cell proliferation, and ultimately alleviating pathological synovial hyperplasia.

Arthritis, Rheumatoid↗
PubMed2004

Expression levels for many genes in human peripheral blood cells are highly sensitive to ex vivo incubation.

Monitoring of gene and protein expression in peripheral blood cells has significant potential for improving the diagnosis and therapy of many human diseases. As genomic-scale microarray and proteomic technologies are applied to peripheral blood, it is important to consider the variables that may affect interpretation of data. Here we report experiments performed to identify genes that are particularly sensitive to ex vivo handling prior to RNA extraction for gene expression microarrays or quantitative real-time RT-PCR assays. We examined Affymetrix gene expression in samples from eight normal individuals where blood was processed for RNA either immediately after blood draw or the next day following overnight incubation. These studies identified hundreds of genes that are sensitive to ex vivo handling of blood, and suggest that this is an important variable to consider when designing and interpreting human PBMC experiments.

Blood Specimen Collection↗
PubMed2003

Absolute quantification of proteins and phosphoproteins from cell lysates by tandem MS.

A need exists for technologies that permit the direct quantification of differences in protein and posttranslationally modified protein expression levels. Here we present a strategy for the absolute quantification (termed AQUA) of proteins and their modification states. Peptides are synthesized with incorporated stable isotopes as ideal internal standards to mimic native peptides formed by proteolysis. These synthetic peptides can also be prepared with covalent modifications (e.g., phosphorylation, methylation, acetylation, etc.) that are chemically identical to naturally occurring posttranslational modifications. Such AQUA internal standard peptides are then used to precisely and quantitatively measure the absolute levels of proteins and posttranslationally modified proteins after proteolysis by using a selected reaction monitoring analysis in a tandem mass spectrometer. In the present work, the AQUA strategy was used to (i) quantify low abundance yeast proteins involved in gene silencing, (ii) quantitatively determine the cell cycle-dependent phosphorylation of Ser-1126 of human separase protein, and (iii) identify kinases capable of phosphorylating Ser-1501 of separase in an in vitro kinase assay. The methods described here represent focused, alternative approaches for studying the dynamically changing proteome.

Amino Acid Sequence↗
PubMed2004

Human T cell lymphotropic virus (HTLV) type-1-specific CD8+ T cells: frequency and immunodominance hierarchy.

Human T cell lymphotropic virus type 1 (HTLV-1) causes HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). We used interferon- gamma enzyme-linked immunospot assays with overlapping peptides spanning the entire HTLV-1 proteome to test whether the HTLV-1-specific CD8(+) T cells differed significantly in frequency or immunodominance hierarchy between patients with HAM/TSP and asymptomatic carriers and whether the frequency correlated with provirus load. Tax was the immunodominant target antigen. There was no significant qualitative or quantitative difference in the HTLV-1-specific CD8(+) T cell response between the 2 groups. Virus-specific CD8(+) T cell frequency alone does not indicate the effectiveness of the cytotoxic T lymphocyte response in controlling provirus load at equilibrium.

CD8-Positive T-Lymphocytes↗
PubMed2005

Progress and new directions in genetics of tuberculosis: an NHLBI working group report.

Tuberculosis (TB), along with AIDS and malaria, is one of the three major killers among infectious diseases. New approaches to preventing, diagnosing, and curing TB are needed, which depend on a better understanding of Mycobacterium tuberculosis and the host. The National Heart, Lung, and Blood Institute convened a working group to develop recommendations for future TB research, including genetic aspects of the disease. The following areas were identified: (1) animal model research to improve understanding of persistence, reactivation, and granulomatous reactions; (2) preclinical studies aimed at shortening treatment of TB; (3) new resources for manipulating and characterizing the M. tuberculosis genome, proteome chips for more specific diagnoses, and studies of genes that appear to be essential but whose functions are not known; (4) prospective studies associated with clinical trials in populations with or at risk of TB to advance development of diagnostics and prognostics; (5) new quantitative and bioinformatic approaches to study the interaction between M. tuberculosis and the infected host and how this influences the infection process; (6) molecular characterization of M. tuberculosis genome diversity and phylogenetic analysis; (7) coordinated studies of human genome scans; (8) genetic epidemiology studies; (9) activities to foster knowledge dissemination, education, and training; and (10) coordination between the National Institutes of Health, the Gates Foundation, the Global Alliance for Tuberculosis Drug Development, and other organizations.

Animals↗
PubMed2006

Finding function: evaluation methods for functional genomic data.

BACKGROUND: Accurate evaluation of the quality of genomic or proteomic data and computational methods is vital to our ability to use them for formulating novel biological hypotheses and directing further experiments. There is currently no standard approach to evaluation in functional genomics. Our analysis of existing approaches shows that they are inconsistent and contain substantial functional biases that render the resulting evaluations misleading both quantitatively and qualitatively. These problems make it essentially impossible to compare computational methods or large-scale experimental datasets and also result in conclusions that generalize poorly in most biological applications. RESULTS: We reveal issues with current evaluation methods here and suggest new approaches to evaluation that facilitate accurate and representative characterization of genomic methods and data. Specifically, we describe a functional genomics gold standard based on curation by expert biologists and demonstrate its use as an effective means of evaluation of genomic approaches. Our evaluation framework and gold standard are freely available to the community through our website. CONCLUSION: Proper methods for evaluating genomic data and computational approaches will determine how much we, as a community, are able to learn from the wealth of available data. We propose one possible solution to this problem here but emphasize that this topic warrants broader community discussion.

Algorithms↗