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At least 955 records · Page 53Linked to original sources
PubMed2003

New molecular research technologies in the study of muscle disease.

PURPOSE OF REVIEW: To describe the current technologies and progress in DNA polymorphism association studies, mRNA expression profiling (microarrays), and proteomics with respect to muscle disease, and the increasing impact of public-access databases of genome-wide information. RECENT FINDINGS: mRNA expression profiling is becoming the most mature of the highly parallel molecular technologies, with microarrays now able to query the large majority of all genes using 1 million oligonucleotide probes built on 1.2-cm2 glass substrates. Applications of microarrays to normal muscle physiology and muscle disease are discussed. Single nucleotide polymorphism association studies promise to determine the predisposition of individuals to acquired muscle disease, including sarcopenia and atrophy, although such studies are in their infancy. Proteomics technologies do not enjoy the sensitivity and specificity of hybridization, and must instead rely on mass spectrometers. Mass spectrometry technology is advancing rapidly, although the sensitivity and throughput is far behind that of mRNA expression profiling. SUMMARY: As the gene mutations responsible for many types of muscular dystrophy and myopathy have been discovered, protein and gene testing has been integrated into the standard patient diagnostic workup. Future developments will include simpler and less expensive molecular diagnostics, advances in the understanding of downstream consequences of these defects, and the genetic predispositions underlying acquired muscle disease.

Female↗
PubMed2006

A tandem affinity tag for two-step purification under fully denaturing conditions: application in ubiquitin profiling and protein complex identification combined with in vivocross-linking.

Tandem affinity strategies reach exceptional protein purification grades and have considerably improved the outcome of mass spectrometry-based proteomic experiments. However, current tandem affinity tags are incompatible with two-step purification under fully denaturing conditions. Such stringent purification conditions are desirable for mass spectrometric analyses of protein modifications as they result in maximal preservation of posttranslational modifications. Here we describe the histidine-biotin (HB) tag, a new tandem affinity tag for two-step purification under denaturing conditions. The HB tag consists of a hexahistidine tag and a bacterially derived in vivo biotinylation signal peptide that induces efficient biotin attachment to the HB tag in yeast and mammalian cells. HB-tagged proteins can be sequentially purified under fully denaturing conditions, such as 8 m urea, by Ni(2+) chelate chromatography and binding to streptavidin resins. The stringent separation conditions compatible with the HB tag prevent loss of protein modifications, and the high purification grade achieved by the tandem affinity strategy facilitates mass spectrometric analysis of posttranslational modifications. Ubiquitination is a particularly sensitive protein modification that is rapidly lost during purification under native conditions due to ubiquitin hydrolase activity. The HB tag is ideal to study ubiquitination because the denaturing conditions inhibit hydrolase activity, and the tandem affinity strategy greatly reduces nonspecific background. We tested the HB tag in proteome-wide ubiquitin profiling experiments in yeast and identified a number of known ubiquitinated proteins as well as so far unidentified candidate ubiquitination targets. In addition, the stringent purification conditions compatible with the HB tag allow effective mass spectrometric identification of in vivo cross-linked protein complexes, thereby expanding proteomic analyses to the description of weakly or transiently associated protein complexes.

Affinity Labels↗
PubMed2026

Mapping the plasma proteomic architecture of systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a heterogeneous systemic autoimmune disease, yet the molecular basis underlying this variability remains incompletely understood. We profiled the plasma proteome in 260 SLE patients and 86 healthy volunteers (HVs) using the SomaScan v4.1 platform, quantifying 7,288 analytes corresponding to 6,595 unique proteins. We identified 215 proteins that were robustly differentially abundant between SLE patients and HVs in both discovery (n = 207 SLE, n = 45 HVs) and validation sets (n = 53 SLE, n = 41 HVs). Within-cases analyses identified 421 proteins associated with disease activity. Network-based clustering delineated correlated protein modules, including an interferon-associated (IFN-associated) module and a kidney-associated module. Autoantibody-stratified analyses further uncovered distinct proteomic endotypes; positivity for antibodies targeting RNA-binding proteins (anti-Sm, anti-Ro-60, anti-RNP68, anti-RNP-A) was associated with increased IFN-stimulated protein levels (e.g., MX1, ISG15, and CXCL10), independent of disease activity. Anti-Sm, anti-RNP-A, and anti-Ro52 antibodies were associated with reduced plasma levels of their respective autoantigens. Anti-dsDNA antibodies were associated with elevated levels of CD40 ligand (CD40LG) and the neutrophil protease, proteinase-3. Moreover, we identified an association between CD40LG and disease activity specific to the anti-dsDNA-positive subgroup. Together, these data define plasma protein signatures of SLE and disease activity, highlight autoantibody-specific molecular phenotypes, and provide a basis for precision medicine.

Humans↗
PubMedDate not supplied

Clinical proteomics: from biomarker discovery and cell signaling profiles to individualized personal therapy.

The discovery of new highly sensitive and specific biomarkers for early disease detection and risk stratification coupled with the development of personalized "designer" therapies holds the key to future treatment of complex diseases such as cancer. Mounting evidence confirms that the low molecular weight (LMW) range of the circulatory proteome contains a rich source of information that may be able to detect early stage disease and stratify risk. Current mass spectrometry (MS) platforms can generate a rapid and high resolution portrait of the LMW proteome. Emerging novel nanotechnology strategies to amplify and harvest these LMW biomarkers in vivo or ex vivo will greatly enhance our ability to discover and characterize molecules for early disease detection, subclassification and prognostic capability of current proteomics modalities. Ultimately genetic mutations giving rise to disease are played out and manifested on a protein level, involving derangements in protein function and information flow within diseased cells and the interconnected tissue microenvironment. Newly developed highly sensitive, specific and linearly dynamic reverse phase protein microarray systems are now able to generate circuit maps of information flow through phosphoprotein networks of pure populations of microdissected tumor cells obtained from patient biopsies. We postulate that this type of enabling technology will provide the foundation for the development of individualized combinatorial therapies of molecular inhibitors to target tumor-specific deranged pathways regulating key biologic processes including proliferation, differentiation, apoptosis, immunity and metastasis. Hence future therapies will be tailored to the specific deranged molecular circuitry of an individual patient's disease. The successful transition of these groundbreaking proteomic technologies from research tools to integrated clinical diagnostic platforms will require ongoing continued development, and optimization with rigorous standardization development and quality control procedures.

Biomarkers↗
PubMed2006

Translational proteomics: developing a predictive capacity -- a review.

Over the past decade, proteomics has undergone a rapid development and radiation, diversifying across the biochemical landscape. While no single technique yet delivers complete proteomic coverage, application-specific adaptations afford significant opportunity for discovery and the development of predictive capacity (e.g. surrogate biomarker and clinical diagnostics). Targeted proteomic approaches, protein profiling strategies using affinity capture mass spectrometry and solution array represent realistic opportunities to deliver predictive capacity. The aim of this review is to provide an overview of proteomic technologies and how the outcomes delivered by such platforms may be translated into applications of predictive utility in clinical and basic science. In particular, recent applications in protein/peptide profiling (solid-phase affinity capture mass spectrometry and the targeted approach of antibody arrays) and the opportunities they afford researchers within the discipline of reproductive biology to develop new diagnostic and prognostic tests and surrogate biomarkers to improve the delivery of women's health care are considered.

Animals↗
PubMed2004

Proteomics in cancer screening and management in gynecologic cancer.

Biomarkers are used routinely for population screening, disease diagnosis and prognosis, monitoring of therapy, and prediction of therapeutic response. Unfortunately, most biomarkers have low sensitivity and specificity and little predictive value. Novel techniques for better screening and early diagnosis of ovarian cancer are urgently needed. Proteomics, the study of the cellular proteins and their activation states, integrates some fundamental techniques, including high-throughput protein purification and profiling, genomic and proteomic databases, and mass spectrometry. In oncology, proteomics will contribute greatly to our understanding of gene functions in tumor development and provide information in clinical applications. This article reviews proteomic techniques and their potential applications in gynecologic cancer screening and management.

Biomarkers, Tumor↗
PubMed2005

Proteomic study of human hepatocellular carcinoma using two-dimensional difference gel electrophoresis with saturation cysteine dye.

To identify the proteomic alterations associated with carcinogenesis of hepatocellular carcinoma (HCC), we compared the protein expression profiles of nine HCC cell lines with those of primary cultured hepatocytes established from five individuals. A differential proteomic study was performed by two-dimensional difference gel electrophoresis, in which protein samples are labeled with different fluorescent dyes and separated according to their isoelectric point and molecular weight. To label the protein samples, we used a newly developed and highly sensitive fluorescent dye, which reacts with all reduced cysteine residues of proteins. Principal component analysis based on the intensity of 1238 protein spots indicated that the HCC cells and the normal hepatocytes had distinct proteomic profiles. The Wilcoxon test was used to determine the protein spots whose intensity was differentially regulated in the HCC cells compared with the normal hepatocytes, and mass spectrometric analysis was used to identify the proteins corresponding to the spots. The proteins identified are involved in cell cycle regulation, binding to a tumor-suppressor gene product, fatty acid binding, and regulation of translation. Western blotting with specific antibodies revealed the overexpression of PCNA, EB1 and E-FABP in HCC tissues compared with noncancerous tissues. Aberrant regulation of EB1 and E-FABP has not previously been implicated in the development of HCC.

Adult↗
PubMed2006

Profiling at mRNA, protein, and metabolite levels reveals alterations in renal amino acid handling and glutathione metabolism in kidney tissue of Pept2-/- mice.

PEPT2 is an integral membrane protein in the apical membrane of renal epithelial cells that operates as a rheogenic transporter for di- and tripeptides and structurally related drugs. Its prime role is thought to be the reabsorption of filtered di- and tripeptides contributing to amino acid homeostasis. To elucidate the role of PEPT2 in renal amino acid metabolism we submitted kidney tissues of wild-type and a Pept2(-/-) mouse line to a comprehensive transcriptome, proteome and metabolome profiling and analyzed urinary amino acids and dipeptides. cDNA microarray analysis identified 147 differentially expressed transcripts in transporter-deficient animals, and proteome analysis by 2D-PAGE and MALDI-TOF-MS identified 37 differentially expressed proteins. Metabolite profiling by GC-MS revealed predominantly altered concentrations of amino acids and derivatives. Urinary excretion of amino acids demonstrated increased glycine and cysteine/cystine concentrations and dipeptides in urine were assessed by amino acid analysis of urine samples before and after in vitro dipeptidase digestion. Dipeptides constituted a noticeable fraction of urinary amino acids in Pept2(-/-) animals, only, and dipeptide-bound glycine and cystine were selectively increased in Pept2(-/-) urine samples. These findings were confirmed by a drastically increased excretion of cysteinyl-glycine (cys-gly). Urinary loss of cys-gly together with lower concentrations of cysteine, glycine, and oxoproline in kidney tissue and altered expression of mRNA and proteins involved in glutathione (GSH) metabolism suggests that PEPT2 is predominantly a system for reabsorption of cys-gly originating from GSH break-down, thus contributing to resynthesis of GSH.

Amino Acids↗
PubMed2007

A combined dataset of human cerebrospinal fluid proteins identified by multi-dimensional chromatography and tandem mass spectrometry.

Human cerebrospinal fluid (CSF) is an important source for studying protein biomarkers of age-related neurodegenerative diseases. Before characterizing biomarkers unique to each disease, it is necessary to categorize CSF proteins systematically and extensively. However, the enormous complexity, great dynamic range of protein concentrations, and tremendous protein heterogeneity due to post-translational modification of CSF create significant challenges to the existing proteomics technologies for an in-depth, nonbiased profiling of the human CSF proteome. To circumvent these difficulties, in the last few years, we have utilized several different separation methodologies and mass spectrometric platforms that greatly enhanced the identification coverage and the depth of protein profiling of CSF to characterize CSF proteome. In total, 2594 proteins were identified in well-characterized pooled human CSF samples using stringent proteomics criteria. This report summarizes our efforts to comprehensively characterize the human CSF proteome to date.

Cerebrospinal Fluid Proteins↗
PubMed2026

Harnessing metabolomics and proteomics in a clinical trial for pulmonary arterial hypertension: insights from post-hoc analysis of the REHAB-PH trial.

BACKGROUND: The significant clinical and molecular heterogeneity of pulmonary arterial hypertension (PAH) poses challenges in identifying effective therapies. Advanced multidimensional profiling offers an opportunity to capture molecular responses and assess biomarker stability, yet its application in randomised trials remains limited. METHODS: We evaluated the multi-omic profiles of participants with PAH in a randomised, placebo-controlled trial of famotidine. Plasma metabolomic and proteomic profiling was performed at enrolment and 24 weeks. Baseline profiles were compared between treatment arms to assess randomisation balance. Intraclass correlation coefficients quantified within-subject stability over time. Linear regression models adjusting for age, sex, body mass index and PAH aetiology evaluated famotidine's molecular effects. False discovery rate was controlled for multiple comparisons. FINDINGS: For the 79 participants, baseline multi-omic profiles were similar between groups. At 24 weeks, 34 and 37 participants remained in the famotidine and placebo groups respectively. The placebo group showed high molecular stability, while greater variability was observed in the famotidine group. Famotidine treatment was associated with significant changes across 191 proteomic pathways (q-value <0.05), but no metabolomic changes remained significant after multiple-testing correction. INTERPRETATION: Integrating multi-omics into a prospective clinical trial is feasible and yields stable longitudinal profiles in the absence of intervention. While famotidine did not yield clinical benefit, associated proteomic changes illustrate how molecular profiling can reveal treatment-related biology and inform future trial design. These findings highlight the broader utility of multi-omics for evaluating drug responses and identifying molecular endotypes in PAH and beyond. FUNDING: US National Institutes of Health.

Humans↗
PubMed2003

Proteomic analyses in Waldenstrom's macroglobulinemia and other plasma cell dyscrasias.

The proteomic analysis of tumor cells emerges as a key complement to gene expression profiling, primarily because regulation of protein expression (at the translational and post-translational levels) can buffer the magnitude of changes occurring at the gene transcription level, in order to fine tune cellular functions. Herein we describe the concept of proteomic analysis of the signaling state of tumor cells, as well as its application in the study of signaling pathways in plasma cell dyscrasias, such as Waldenstrom's macroglobulinemia (WM) and multiple myeloma (MM). Comparative studies of WM versus MM cells at baseline and in the setting of drug treatment reveals proteomic profiles of the signaling state with significant overlap (that could reflect a putative B-cell lineage-related proteomic signature), but also distinct differences, possibly associated with differential features in the biologic behavior and drug sensitivity of these diseases. These proteomic studies pave the way for a more comprehensive insight into the molecular basis of WM versus other B-cell malignancies.

Electrophoresis, Gel, Two-Dimensional↗
PubMed2001

Novel method for the proteomic investigation of a dairy-associated Bacillus cereus biofilm.

The biofilm proteome of a dairy-associated Bacillus cereus strain (B. cereus 5) was investigated. Biofilm biomass of sufficient concentration for 2D-PAGE was obtained by growing the culture in the presence of glass wool. B. cereus 5 readily attached to the glass wool and biofilms formed within 18 h. The biofilm proteome of whole-cell proteins revealed that 10 proteins were synthesized as a result of surface attachment of which four were unique to the biofilm profile. Seven proteins appeared to be absent in the biofilm profile. The altered proteomes indicated that changes took place in the regulation of protein expression when B. cereus 5 cells attached to surfaces.

Bacillus cereus↗
PubMed2004

Multimembrane dot-blotting: a cost-effective tool for proteome analysis.

The molecular profiles of protein expression from hundreds of cell lysates can be determined in a high-throughput manner by using fluorescent bead technologies, enzyme-linked immunosorbent assays (ELISAs), and protein microarrays. Although powerful, these tools are costly and technically challenging and thus have limited accessibility for many research groups. We propose a modification of traditional dot blotting that increases throughput of this approach and provides a simple and cost-effective technique for profiling multiple samples. In contrast to traditional blotting that uses a single membrane, we introduce blotting onto a stack of novel, thin, sieve-like membranes. These membranes have a high affinity for binding proteins, but have a lower capacity of protein binding compared to traditional (nitrocellulose) membranes. We compare the linear binding capacity and variability of these novel membranes with nitrocellulose membranes. Also, we describe the use of these membranes in a multilayer dot blot format for profiling mitogen-mediated signal transduction pathways in T cells.

Blood Proteins↗
PubMed2003

Proteomic analysis of hematopoietic stem cell-like fractions in leukemic disorders.

DNA microarray analysis has been applied to identify molecular markers of human hematological malignancies. However, the relatively low correlation between the abundance of a given mRNA and that of the encoded protein makes it important to characterize the protein profile directly, or 'proteome,' of malignant cells in addition to the 'transcriptome.' To identify proteins specifically expressed in leukemias, here we isolated AC133(+) hematopoietic stem cell-like fractions from the bone marrow of 13 individuals with various leukemic disorders, and compared their protein profiles by two-dimensional electrophoresis. A total of 11 differentially expressed protein spots corresponding to 10 independent proteins were detected, and peptide fingerprinting combined with mass spectrometry of these proteins revealed them to include NuMA (nuclear protein that associates with the mitotic apparatus), heat shock proteins, and redox regulators. The abundance of NuMA in the leukemic blasts was significantly related to the presence of complex karyotype anomalies. Conditional expression of NuMA in a mouse myeloid cell line resulted in the induction of aneuploidy, cell cycle arrest in G(2)-M phases, and apoptosis. These results demonstrate the potential of proteome analysis with background-matched cell fractions obtained from fresh clinical specimens to provide insight into the mechanism of human leukemogenesis.

Adult↗
PubMed2006

Morphine administration alters the profile of hippocampal postsynaptic density-associated proteins: a proteomics study focusing on endocytic proteins.

Numerous studies have shown that drugs of abuse induce changes in protein expression in the brain that are thought to play a role in synaptic plasticity. Drug-induced plasticity can be mediated by changes at the synapse and more specifically at the postsynaptic density (PSD), which receives and transduces synaptic information. To date, the majority of studies examining synaptic protein profiles have focused on identifying the synaptic proteome. Only a handful of studies have examined the changes in synaptic profile by drug administration. We applied a quantitative proteomics analysis technique with the cleavable ICAT reagent to quantitate relative changes in protein levels of the hippocampal PSD in response to morphine administration. We identified a total of 102 proteins in the mouse hippocampal PSD. The majority of these were signaling, trafficking, and cytoskeletal proteins involved in synaptic plasticity, learning, and memory. Among the proteins whose levels were found to be altered by morphine administration, clathrin levels were increased to the largest extent. Immunoblotting and electron microscopy studies showed that this increase was localized to the PSD. Morphine treatment was also found to lead to a local increase in two other components of the endocytic machinery, dynamin and AP-2, suggesting a critical involvement of the endocytic machinery in the modulatory effects of morphine. Because alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors are thought to undergo clathrin-mediated endocytosis, we examined the effect of morphine administration on the association of the AMPA receptor subunit, GluR1, with clathrin. We found a substantial decrease in the levels of GluR1 associated with clathrin. Taken together, these results suggest that, by causing a redistribution of endocytic proteins at the synapse, morphine modulates synaptic plasticity at hippocampal glutamatergic synapses.

Amino Acid Sequence↗
PubMed2004

Comparative proteomic analysis of de-N-glycosylated serum from hepatitis B carriers reveals polypeptides that correlate with disease status.

Analysis of the polypeptide profile in tissues, cells, and sera by high-resolution two-dimensional (2-D) gel electrophoresis offers promise in the identification of biomarkers that correlate with disease. However, sera contain many polypeptides bearing N-linked glycosylation that can complicate interpretation. Therefore, we tested the possibility that de-N-glycosylation of the polypeptides present in human serum would result in a simplification of serum proteome profiles. Briefly, polypeptides present in human serum were left untreated or subjected to de-N-glycosylation by incubation with PNGase F and resolved by high-resolution 2-D gel electrophoresis. De-N-glycosylation reduced the number of glycoform variants, enhanced the resolution of many polypeptides and allowed other polypeptides to become visible. As an initial test of concept, clinically relevant serum samples from individuals with or without diagnosis of hepatocellular carcinoma were compared. Several polypeptides, apparent only after de-N-glycosylation, were shown to correlate with disease. Although the results are preliminary and the identities of all the putative biomarkers not yet known, the data suggest that de-N-glycosylation offers a method to enhance the resolution of serum polypeptide profiles and has value in comparative proteomic studies.

Amyloid↗
PubMed2007

Proteomics analysis in lung cancer: challenges and opportunities.

Recent technological developments in proteomic analysis are bringing us new insights into the molecular classification of tumours. Although proteomic analysis in cancer profiling is still under development both in terms of the instruments used and the data analytical tools, this method has great potential advantages for the analysis of biospecimens of many types. Direct measurement of abnormally expressed or modified proteins in the tumour tissue and/or patient blood may be an effective approach for discovering new biomarkers. Proteomics has the significant advantage of being able to discern not only changes in expression levels but also in post-translational modifications. Thus, the proteomics approach to protein profiling and biomarker discovery uncovers biomarkers from a different viewpoint than microarray analysis. This review summarizes the range of proteomics technologies employed for cancer profiling, and how they have been used to derive new classification models for human lung cancer.

Biomarkers, Tumor↗
PubMed2006

Comparison of extraction procedures for proteome analysis of Streptococcus pneumoniae and a basic reference map.

Streptococcus pneumoniae is an important human pathogen causing life-threatening invasive diseases such as pneumonia, meningitis and bacteraemia. Despite major advances in our understanding of pneumococcal mechanisms of pathogenicity obtained through genomic studies very little has been achieved on the characterisation of the proteome of this pathogen. The highly complex structure of its cell envelope particularly amongst the various capsular forms enables the cell to resist lysis by conventional mechanical methods. It is therefore highly desirable to develop a cellular lysis and protein solubilisation procedure that minimises protein losses and allows for maximum possible coverage of the proteome of S. pneumoniae. Here we have utilised various combinations of mechanical or enzymatic cell lysis with two protein solubilisation mixtures urea/CHAPS-based mixture or SDS/DTT-based mixture in order to achieve best quality protein profiles using two proteomic technologies surface-enhanced laser desorption ionisation (SELDI) TOF MS and 2-DE. While urea/CHAPS-based mixture combined with freeze/thawing provided enough material for good-quality SELDI TOF MS fingerprints, a combination of mechanical, enzymatic and chemical lysis was needed to be used to successfully extract the desired protein content for 2-DE analysis. The methods chosen were also assessed for reproducibility and tested on various capsular types of S. pneumoniae. As a result, good-quality and reproducible profiles were created using various ProteinChip arrays and more than 800 protein spots were separated on a single 2-D gel of S. pneumoniae. Twenty-five of the most abundant protein spots were identified using LC/MS/MS to create a reference map of S. pneumoniae. The proteins identified included glycolytic enzymes such as glyceraldehyde 3-phosphate dehydrogenase, phosphoglycerate kinase, enolase etc. Several fermentation enzymes were also present including two of the components of the arginine deiminase system. Proteins involved in protein synthesis, such as translation factors and ribosomal proteins, as well as several chaperone proteins were also identified.

Amino Acid Sequence↗