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PubMed2006

Two-dimensional difference gel electrophoresis.

The two-dimensional (2D) polyacrylamide gel-based approach to protein profiling has been successful because it is an accessible, inexpensive, and powerful tool for the analysis of global patterns of protein expression. All protein spots that are resolved and detected within the 104 to 105 dynamic range of gel capacity can be studied qualitatively and quantitatively in relation to each other, and viewed as a single image. Two-dimensional difference gel electrophoresis (DIGE) has strengthened the 2D platform by allowing the detection and quantitation of differences between samples resolved on the same gel, or across multiple gels, when linked by an internal standard. The technology is based on modification of protein extracts with fluorescent cyanine dyes, which have distinct excitation and emission spectra and are migration (charge and size) matched so that the same protein labeled with any of the dyes (Cy2, Cy3, Cy5) will migrate to the same position within a 2D gel, greatly enhancing reproducibility. It is becoming clear that each technology that is currently available for quantifying differential protein expression has its own weaknesses and strengths, and that multiplatform, integrated approaches will be necessary to provide the most complete analysis of any given proteome. We believe that DIGE is, and will remain in the future, a key front-end proteomic tool that will strongly complement other protein-profiling technologies.

Animals↗
PubMed2003

The quest for the mechanisms of life.

The genomic revolution, manifested by the sequencing of the complete genome of many organisms, along with technological advances, such as DNA microarrays and developments in high-throughput analysis of proteins, metabolites, and isotopic tracer distribution patterns, challenged the conventional ways in which questions are approached in the biological sciences: (a) rather than examining a small number of genes and/or reactions at any one time;, we can now analyze gene expression and protein activity in the context of systems of interacting genes and gene products; (b) comprehensive analysis of biological systems requires the integration of all cellular fingerprints: genome sequence, maps of gene expression, protein expression, metabolic output, and in vivo enzymatic activity; and (c) collecting, managing, and analyzing comparable data from various cellular profiles requires expertise from several fields that transcend traditional discipline boundaries. While researchers in systems biology have still to address difficult challenges in both experimental and computational arenas, they possess, for the first time, the opportunity to unravel the mechanisms of life. The enormous impact of these discoveries in diverse areas, such as metabolic engineering, strain selection, drug screening and development, bioprocess development, disease prognosis and diagnosis, gene and other medical therapies, is an obvious motivation for pursuing integrated analyses of cellular systems.

Cell Physiological Phenomena↗
PubMed2001

Gene induction during differentiation of human monocytes into dendritic cells: an integrated study at the RNA and protein levels.

Changes in gene expression occurring during differentiation of human monocytes into dendritic cells were studied at the RNA and protein levels. These studies showed the induction of several gene classes corresponding to various biological functions. These functions encompass antigen processing and presentation, cytoskeleton, cell signalling and signal transduction, but also an increase in mitochondrial function and in the protein synthesis machinery, including some, but not all, chaperones. These changes put in perspective the events occurring during this differentiation process. On a more technical point, it appears that the studies carried out at the RNA and protein levels are highly complementary.

Cell Differentiation↗
PubMed2006

Early legume responses to inoculation with Rhizobium sp. NGR234.

Interactions between legumes and rhizobia are controlled by the sequential exchange of symbiotic signals. Two different techniques, 2D-PAGE electrophoresis and differential display were used to study the effects of rhizobial signals on legume development. Application of variously substituted lipo-oligo-saccharidic Nod-factors to roots of Vigna unguiculata resulted in changes in the phosphorylation patterns of microsomal proteins. Reliable amino-acid sequences were obtained for one Nod-factor enhanced protein which was highly homologous to the 57-kDa subunit from Arabidopsis thaliana vacuolar membrane H(+)-ATPase. Immuno-blotting techniques demonstrated that Nod-factors cause rapid and massive increases of this enzyme in treated roots, suggesting that H(+)-ATPases play symbiotic roles. Concomitantly, we used differential display (DD) techniques on mRNA isolated from root-hairs to analyse early root responses to NGR234. Significant matches of several DD clones to known sequences were found. Clone D2.62 was homologous to a multitude of receptor kinases including S receptor-like kinases of A. thaliana and clone D4.1 showed similarities to Lotus japonicus phosphatidylinositol transfer-like protein III and late nodulin 16. Independent confirmatory analyses of these differentially expressed clones indicated expression at very low levels.

Cell Fractionation↗
PubMed2002

Muscular dystrophy into the new millennium.

Since the identification of the gene for Duchenne muscular dystrophy and its protein product some 15 years ago, the basic defects in all the commoner forms of dystrophy have now been identified. It is thus possible, on the basis of this information, to make a precise diagnosis in an affected individual and to offer accurate genetic counselling and prenatal diagnosis. Now newer technologies are being applied to the investigation of these disorders. These include studies of single nucleotide polymorphisms, microarray analysis and expression profiling, the yeast two-hybrid assay, and proteomics. A great deal of new information is emerging in this way which will hopefully help us to understand the causes of inter-familial and intra-familial variation and particularly pathogenesis, a detailed understanding of which could be the first step in finding effective treatments.

Humans↗
PubMed2003

Advancing drug discovery through systems biology.

Pharmaceutical companies are facing an urgent need to both increase their lead compound and clinical candidate portfolios and satisfy market demands for continued innovation and revenue growth. Here, we outline an emerging approach that attempts to facilitate and alleviate many of the current drug discovery issues and problems. This is, in part, achieved through the systematic integration of technologies, which results in a superior output of data and information, thereby enhancing our understanding of biological function, chemico-biological interactions and, ultimately, drug discovery. Systems biology is one new discipline that is positioned to significantly impact this process.

Animals↗
PubMed2006

Effect of 5-azacytidine on the protein expression of porcine bone marrow mesenchymal stem cells in vitro.

Bone marrow-derived mesenchymal stem cells (MSCs) are pluripotent stem cells that show a vital potential in the clinical application for cell transplantation. In the present paper, proteomic techniques were used to approach the protein profiles associated with porcine bone marrow MSCs and investigate the regulation of MSC proteins on the effect of 5-azacytidine (5-aza). Over 1,700 protein species were separated from MSCs according to gel analysis. Compared with the expression profiling of control MSCs, there were 11 protein spots up-regulated and 26 down-regulated in the protein pattern of 5-aza-treated cells. A total of 21 proteins were successfully identified by MALDI-TOF-MS analysis, among which some interesting proteins, such as alpha B-crystallin, annexin A2, and stathmin 1, had been reported to involve in cell proliferation and differentiation through different signaling pathways. Our data should be useful for the future study of MSC differentiation and apoptosis.

Animals↗
PubMedDate not supplied

Identification of protein fragments as pattern features in MALDI-MS analyses of serum.

The use of matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) to acquire spectral profiles has become a common approach to detect proteomic biomarkers of disease. MALDI-MS signals may represent both intact proteins as well as proteolysis products. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis can tentatively identify the corresponding proteins Here, we describe the application of a data analysis utility called FragMint, which combines MALDI-MS spectral data with LC-MS/MS based protein identifications to generate candidate protein fragments consistent with both types of data. This approach was used to identify protein fragments corresponding to spectral signals in MALDI-MS analyses of unfractionated human serum. The serum also was analyzed by one-dimensional SDS-PAGE and bands corresponding to the MALDI-MS signal masses were excised and subjected to in-gel digestion and LC-MS/MS analysis. Database searches mapped all of the identified peptides to abundant blood proteins larger than the observed MALDI-MS signals. FragMint identified fragments of these proteins that contained the MS/MS identified sequences and were consistent with the observed MALDI-MS signals. This approach should be generally applicable to identify protein species corresponding to MALDI-MS signals.

Algorithms↗
PubMed2004

Antimicrobial drug discovery through bacteriophage genomics.

Over evolutionary time bacteriophages have developed unique proteins that arrest critical cellular processes to commit bacterial host metabolism to phage reproduction. Here, we apply this concept of phage-mediated bacterial growth inhibition to antibiotic discovery. We sequenced 26 Staphylococcus aureus phages and identified 31 novel polypeptide families that inhibited growth upon expression in S. aureus. The cellular targets for some of these polypeptides were identified and several were shown to be essential components of the host DNA replication and transcription machineries. The interaction between a prototypic pair, ORF104 of phage 77 and DnaI, the putative helicase loader of S. aureus, was then used to screen for small molecule inhibitors. Several compounds were subsequently found to inhibit both bacterial growth and DNA synthesis. Our results suggest that mimicking the growth-inhibitory effect of phage polypeptides by a chemical compound, coupled with the plethora of phages on earth, will yield new antibiotics to combat infectious diseases.

Anti-Bacterial Agents↗
PubMed2005

Identifying regulatory subnetworks for a set of genes.

High throughput genomic/proteomic strategies, such as microarray studies, drug screens, and genetic screens, often produce a list of genes that are believed to be important for one or more reasons. Unfortunately it is often difficult to discern meaningful biological relationships from such lists. This study presents a new bioinformatic approach that can be used to identify regulatory subnetworks for lists of significant genes or proteins. We demonstrate the utility of this approach using an interaction network for yeast constructed from BIND, TRANSFAC, SCPD, and chromatin immunoprecipitation (ChIP)-Chip data bases and lists of genes from well known metabolic pathways or differential expression experiments. The approach accurately rediscovers known regulatory elements of the heat shock response as well as the gluconeogenesis, galactose, glycolysis, and glucose fermentation pathways in yeast. We also find evidence supporting a previous conjecture that approximately half of the enzymes in a metabolic pathway are transcriptionally co-regulated. Finally we demonstrate a previously unknown connection between GAL80 and the diauxic shift in yeast.

Algorithms↗
PubMed2006

Progressive peak clustering in GC-MS Metabolomic experiments applied to Leishmania parasites.

MOTIVATION: A common problem in the emerging field of metabolomics is the consolidation of signal lists derived from metabolic profiling of different cell/tissue/fluid states where a number of replicate experiments was collected on each state. RESULTS: We describe an approach for the consolidation of peak lists based on hierarchical clustering, first within each set of replicate experiments and then between the sets of replicate experiments. The problems of finding the dendrogram tree cutoff which gives the optimal number of peak clusters and the effect of different clustering methods were addressed. When applied to gas chromatography-mass spectrometry metabolic profiling data acquired on Leishmania mexicana, this approach resulted in robust data matrices which completely separated the wild-type and two mutant parasite lines based on their metabolic profile.

Animals↗
PubMed2005

Prediction of protein subcellular locations using a new measure of information discrepancy.

Given a raw protein sequence, knowing its subcellular location is an important step toward understanding its function and designing further experiments. A novel method is proposed for the prediction of protein subcellular locations from sequences. For four categories of eukaryotic proteins the overall predictive accuracy is 82.0%, 2.6% higher than that by using SVM approach. For three subcellular locations of prokaryotic proteins, an overall accuracy of 89.9% is obtained. In accordance with the architecture of cells, a hierarchical prediction approach is designed. Based on amino acid composition extracellular proteins and intracellular proteins can be identified with accuracy of 97%.

Algorithms↗
PubMed2026

Downregulated lysyl oxidase in plasma extracellular vesicles: a biomarker linked to brain metastasis risk in lung adenocarcinoma.

BACKGROUND: Brain metastasis (BrM) is a leading cause of mortality in patients with lung adenocarcinoma (LUAD). Extracellular vesicles (EVs), which carry bioactive molecules, play a critical role in tumor microenvironment remodeling and exhibit metastatic organotropism, holding promise as liquid biopsy biomarkers. This study aims to identify plasma EV-derived proteins associated with LUAD-BrM. METHODS: A multi-omics framework was applied. Plasma EVs from 59 stage IV LUAD patients (30 BrM vs 29 non-BrM) were profiled using data-independent acquisition mass spectrometry proteomics. Candidate proteins were screened via bioinformatics and machine learning (LASSO/RF/SVM). Initial validation included tissue proteomics (n = 13), single-cell transcriptomics (TISCH2), and Western blot analysis of a subset of the discovery samples. Functional experiments were conducted in vitro. The lead candidate was ultimately validated in an independent plasma cohort (n = 158) through ELISA. RESULTS: Proteomic analysis implicated collagen-containing extracellular matrix (ECM) pathways. Lysyl oxidase (LOX), a key ECM cross-linking enzyme, was identified as a lead candidate. LOX and its family member LOXL1 were consistently downregulated in BrM tissues and plasma EVs. Single-cell analysis revealed decreased LOX expression specifically in BrM-associated fibroblasts, which showed suppressed ECM-related pathways. In vitro experiments supported a PI3K/AKT-LOX-ECM regulatory axis. Plasma EV-derived LOX demonstrated strong diagnostic performance in the independent cohort, with an AUC of 0.786 (95% CI 0.713iated fi. CONCLUSIONS: Our study establishes plasma EV-derived LOX as a promising non-invasive biomarker for LUAD-BrM through a comprehensive multi-omics validation strategy. We propose a model wherein downregulation of LOX, potentially driven by PI3K/AKT signaling in tumor-associated fibroblasts, contributes to ECM degradation and may promote brain-tropic metastasis. This finding offers new insights for risk stratification and timely intervention in LUAD patients.

Humans↗
PubMed2005

Optimal molecular profiling of tissue and tissue components: defining the best processing and microdissection methods for biomedical applications.

Isolation of well-preserved pure cell populations is a prerequisite for sound studies of the molecular basis of any tissue-based biological phenomenon. This article reviews current methods for obtaining anatomically specific signals from molecules isolated from tissues, a basic requirement for productive linking of phenotype and genotype. The quality of samples isolated from tissue and used for molecular analysis is often glossed over or omitted from publications, making interpretation and replication of data difficult or impossible. Fortunately, recently developed techniques allow life scientists to better document and control the quality of samples used for a given assay, creating a foundation for improvement in this area. Tissue processing for molecular studies usually involves some or all of the following steps: tissue collection, gross dissection/identification, fixation, processing/embedding, storage/archiving, sectioning, staining, microdissection/annotation, and pure analyte labeling/identification and quantification. We provide a detailed comparison of some current tissue microdissection technologies, and provide detailed example protocols for tissue component handling upstream and downstream from microdissection. We also discuss some of the physical and chemical issues related to optimal tissue processing, and include methods specific to cytology specimens. We encourage each laboratory to use these as a starting point for optimization of their overall process of moving from collected tissue to high quality, appropriately anatomically tagged scientific results. In optimized protocols is a source of inefficiency in current life science research. Improvement in this area will significantly increase life science quality and productivity. The article is divided into introduction, materials, protocols, and notes sections. Because many protocols are covered in each of these sections, information relating to a single protocol is not contiguous. To get the greatest benefit from this article, readers are advised to read through the entire article first, identify protocols appropriate to their laboratory for each step in their workflow, and then reread entries in each section pertaining to each of these single protocols.

Biomarkers↗