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At least 595 records · Page 33Linked to original sources
PubMed2026

Nuclear Proteome Map of Mouse Heart Chambers.

Heart specialization involves nuclear programs; however, chamber-specific regulation of the nuclear proteome landscape remains unknown. In this study, we isolated the nucleus from four major anatomical regions of healthy mouse heart (fresh) and employed quantitative mass spectrometry-based proteomics to construct a comprehensive nuclear proteome landscape of left ventricle (LV, 2403 proteins), right ventricle (RV, 2242 proteins), left atrium (LA, 2368 proteins), and right atrium (RA, 1816 proteins). This led to the discovery of nuclear regional proteome signatures (ventricular signature, 297 proteins; atrial signature, 183 proteins) associated with oxidative metabolism and redox regulation, ferroptosis, extracellular-matrix remodeling, SUMO- and stress-responsive control and transcriptional regulation. Chamber-level analyses further identify distinct nuclear features in LV (120 proteins), LA (188 proteins), and RA (72 proteins). In addition, we defined conserved core nuclear proteome (230 proteins) shared across all anatomical regions, enriched for transcription-regulator complexes, nucleolar/ribosome-associated, RNA-processing, and chromatin-organization components. Within this core network, we report 78 transcription factors/co-factors and select nuclear, chromatin and RNA export-associated proteins, including 29 specific factors (e.g., Alpk3, Rbm14, Arglu1, Hmgb1, Myef2, Sf1) associated with the heart. Regionally, we verified spatial localization in heart of H2ac21 and Sun2 in LA and Ptbp2 in LV by immunofluorescence. This study provides insights into the chamber-resolved view of the nuclear proteome in the heart, establishes a framework for linking nuclear proteomic signatures to atrial and ventricular biology, unique features of the heart nuclear proteome landscape relative to other organs, and a baseline for studying nuclear remodeling in cardiac pathophysiology.

Animals↗
PubMed2006

Identification of over-expressed proteins in oral squamous cell carcinoma (OSCC) patients by clinical proteomic analysis.

BACKGROUND: Oral cancer is a worldwide problem. It is a universal aggressive disease in the population of smoking and drinking. The oral cancer mortality has been ranked 5th place in Taiwan in male cancer patients. A number of protein markers for oral cancer are still not applicable in large populations. Proteomic technologies provide excellent tools for rapid screening of a large number of potential biomarkers in malignant cells. METHOD: Proteomics and real-time quantitative RT-PCR were used to analyze over-expressed proteins in 10 OSCC patients. RESULT: Forty-one proteins were identified as commonly over-expressed in OSCC tissues. In OSCC tissues, alphaB-crystallin, tropomyosin 2, myosin light chain 1, heat shock protein 27 (HSP27), stratifin, thioredoxin-dependent peroxide reductase, flavin reductase, vimentin, rho GDP-dissociation inhibitor 2 (rho GDI-2), glutathione S-transferase Pi (GST-pi) and superoxide dismutase [Mn] (MnSOD) were significantly over-expressed (an average of 7.2, 6.0, 5.7, 4.3, 3.6, 3.4, 3.0, 3.0, 2.6, 2.5, 2.1-fold, respectively). In real-time quantitative RT-PCR analysis, the gene expressions of alphaB-crystallin, HSP27 and MnSOD were also increased in the cancer tissues, consistent with proteomic results. CONCLUSION: The identified proteins in this experiment may be used in future studies of carcinogenesis or as diagnostic markers and therapeutic targets for OSCC.

Adult↗
PubMed2025

Application of Proteomic Methods in Oomycete Biology.

The biochemical makeup of any organism provides insight into key factors regarding its biological functions. These factors can be explored using proteomics, which allows us to obtain a snapshot of the protein content and abundance in an organism, cell type or sub-cellular compartment. Here, we describe proteomic methodologies that can be used to dissect the biochemical mechanism of phytopathogenicity in oomycetes. These methodologies include protein extraction, purification, subsequent processing, mass spectrometry analysis, and qualitative and quantitative data processing of oomycete proteomes for comparative studies. Additionally, the use of mass spectra to assist in gene validation and modelling in unfinished oomycete genomes is also described.

Oomycetes↗
PubMed2006

Application of two-dimensional gel electrophoresis to interrogate alterations in the proteome of genetically modified crops. 1. Assessing analytical validation.

Current tools used to assess the safety of food and feed derived from modern biotechnology emphasize the investigation of possible unintended effects caused directly by the expression of transgenes or indirectly by pleiotropy. These tools include extensive multisite and multiyear agronomic evaluations, compositional analyses, animal nutrition, and classical toxicology evaluations. Because analytical technologies are rapidly developing, proteome analysis based on two-dimensional gel electrophoresis (2DE) was investigated as a complementary tool to the existing technologies. A 2DE method was established for the qualitative and quantitative analysis of the seed proteome of Arabidopsis thaliana with the following validation parameters examined: (1) source and scope of variation; (2) repeatability; (3) sensitivity; and (4) linearity of the method. The 2DE method resolves proteins with isoelectric points between 4 and 9 and molecular masses (MM) of 6-120 kDa and is sensitive enough to detect protein levels in the low nanogram range. The separation of the proteins was demonstrated to be very reliable with relative position variations of 1.7 and 1.1% for the pI and MM directions, respectively. The mean coefficient of variation of 254 matched spot qualities was found to be 24.8% for the gel-to-gel and 26% for the overall variability. A linear relationship (R2 > 0.9) between protein amount and spot volume was demonstrated over a 100-fold range for the majority of selected proteins. Therefore, this method could be used to interrogate proteome alterations such as a novel protein, fusion protein, or any other change that affects molecular mass, isoelectric point, and/or quantity of a protein.

Arabidopsis↗
PubMed2004

High-throughput proteomics using Fourier transform ion cyclotron resonance mass spectrometry.

The advent of high-throughput proteomic technologies for global detection and quantitation of proteins creates new opportunities and challenges for those seeking to gain greater understanding of the cellular machinery. Here, recent advances in high-resolution capillary liquid chromatography coupled to Fourier transform ion cyclotron resonance mass spectrometry are reviewed along with its potential application to high-throughput proteomics. These technological advances combined with quantitative stable isotope labeling methodologies provide powerful tools for expanding our understanding of biology at the system level.

Animals↗
PubMed2006

Advances and challenges in liquid chromatography-mass spectrometry-based proteomics profiling for clinical applications.

Recent advances in proteomics technologies provide tremendous opportunities for biomarker-related clinical applications; however, the distinctive characteristics of human biofluids such as the high dynamic range in protein abundances and extreme complexity of the proteomes present tremendous challenges. In this review we summarize recent advances in LC-MS-based proteomics profiling and its applications in clinical proteomics as well as discuss the major challenges associated with implementing these technologies for more effective candidate biomarker discovery. Developments in immunoaffinity depletion and various fractionation approaches in combination with substantial improvements in LC-MS platforms have enabled the plasma proteome to be profiled with considerably greater dynamic range of coverage, allowing many proteins at low ng/ml levels to be confidently identified. Despite these significant advances and efforts, major challenges associated with the dynamic range of measurements and extent of proteome coverage, confidence of peptide/protein identifications, quantitation accuracy, analysis throughput, and the robustness of present instrumentation must be addressed before a proteomics profiling platform suitable for efficient clinical applications can be routinely implemented.

Biomarkers↗
PubMed2005

Quantitative analysis of cytochrome p450 isozymes by means of unique isozyme-specific tryptic peptides: a proteomic approach.

A novel matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry method has been developed to quantitate cytochrome P450 (P450) isozymes based on their unique isozyme-specific tryptic peptides. It was shown that the molar ratio of P450 isozyme-specific peptides is linearly proportional to the mass peak area ratio of corresponding peptides not only in simple two-peptide mixtures, but also in complex digest mixtures. This approach is applicable both to in-gel (as shown for CYP2B1 and CYP2B2) and in-solution digests (as shown for CYP1A2, CYP2E1, and CYP2C19) and does not require introduction of stable isotopes or labeling with isotope-coded affinity tagging. The relative and absolute quantitation can be performed after developing corresponding calibration curves with synthesized P450 isozyme-specific peptide standards. The absolute quantitation of human P450 isozymes was performed by using CYP2B2 isozyme-specific peptide (1306.7 Da) as the universal internal standard. The utility of this approach was demonstrated for two highly homologous (>97%) rat liver CYP2B1 and CYP2B2 and three human P450 isozymes belonging to two different families and three different subfamilies: CYP1A2, CYP2E1, and CYP2C19. In summary, we have demonstrated that MALDI TOF-based peptide mass fingerprinting of different cytochrome P450 isozymes can provide not only qualitative but quantitative data, too.

Animals↗
PubMed2006

Transcriptome and proteome profiling to understanding the biology of high productivity CHO cells.

A combined transcriptome and proteome analysis was carried out to identify key genes and proteins differentially expressed in Chinese hamster ovary (CHO) cells producing high and low levels of dhfr-GFP fusion protein. Comparison of transcript levels was performed using a proprietary 15K CHO cDNA microarray chip, whereas proteomic analysis was performed using iTRAQ quantitative protein profiling technique. Microarray analysis revealed 77 differentially expressed genes, with 53 genes upregulated and 24 genes downregulated. Proteomic analysis gave 75 and 80 proteins for the midexponential and stationary phase, respectively. Although there was a general lack of correlation between mRNA levels and quantitated protein abundance, results from both datasets concurred on groups of proteins/genes based on functional categorization. A number of genes (20%) and proteins (45 and 23%) were involved in processes related to protein biosynthesis. We also identified three genes/proteins involved in chromatin modification. Enzymes responsible for opening up chromatin, Hmgn3 and Hmgb1, were upregulated whereas enzymes that condense chromatin, histone H1.2, were downregulated. Genes and proteins that promote cell growth (Igfbp4, Ptma, S100a6, and Lgals3) were downregulated, whereas those that deter cell growth (Ccng2, Gsg2, and S100a11) were upregulated. Other main groups of genes and proteins include carbohydrate metabolism, signal transduction, and transport. Our findings show that an integrated genomic and proteomics approach can be effectively utilized to monitor transcriptional and posttranscriptional events of mammalian cells in culture.

Animals↗
PubMed2003

Use of proteomics methodology to evaluate inflammatory protein expression in tendinitis.

In previous studies we established a rat model of acute tendinitis including functional and mechanical measures of healing. Achilles' tendinitis was induced by injection of collagenase, an enzyme that produces localized fiber digestion and edema formation. As quantitative measures of tissue inflammation, hypercellularity and edema were evaluated in injured tendons in comparison with controls. Using the rat tendinitis model, we have applied isotope-coded affinity tag analysis (ICAT) methodology to indicate localized tendon healing by quantitating protein expression. This novel proteomics method allows detection of subtle differences in protein levels that provide a detailed picture of tendinitis healing. The method involves a new class of chemical linkers used to differentially label cysteine residues from similar peptides in control and treated protein samples with heavy (deuterium off of backbone) and light (hydrogen off of backbone) ICAT reagents that are otherwise chemically identical. Proteins were extracted under liquid nitrogen from control untreated or injured Achilles' tendons 72 hours after collagenase-injection. These proteins were digested with endoproteinase Glu-C and trypsin and the resulting peptide mixtures were evaluated using reverse-phase C18 HPLC and Tristricine SDS-polyacrylamide gel electrophoresis. The two ICAT-modified peptide populations were mixed, affinity-purified and analyzed using microcapillary liquid chromatography and electrospray ionization tandem mass-spectroscopy. The process resulted in relative abundance and charge-to-mass ratio data used in conjunction with database searching to identify proteins expressed differentially in the two treatment groups. By analyzing different time periods in the healing process, an accurate model of the healing rat tendon can be made.

Achilles Tendon↗
PubMed2006

Combined chemical and enzymatic stable isotope labeling for quantitative profiling of detergent-insoluble membrane proteins isolated using Triton X-100 and Brij-96.

Effective quantitative profiling of detergent-insoluble membrane proteins using high-throughput mass spectrometry (MS)-based proteomics would allow a better understanding of physiological and pathological processes that take place at the cell surface. To increase the coverage of proteins present in detergent-resistant membrane microdomains (DRMMs), a combination of 16O/18O and isotope coded affinity tags (ICAT) labeling was used in a comparative analysis of detergent-insoluble membrane proteins isolated from rat basophilic leukemia cells (RBL-2H3), with either Triton X-100 or Brij-96. The analysis resulted in the quantification of 738 unique proteins from Triton X-100 and Brij-96 isolated DRMMs, significantly exceeding the number of proteins quantified from either single labeling technique. Twenty-five noncysteine-containing proteins were quantified, as well as 32 cysteine-containing proteins that would have been missed if either 16O/18O or ICAT labeling had been used exclusively, which illustrate better proteome coverage and enhanced ability to quantitate. The comparative analysis revealed that proteins were more readily extracted using Triton X-100 than Brij-96; however, Triton X-100 also extracted larger quantities of non-DRMMs-associated proteins. This result confirms previous, targeted studies suggesting that DRMMs isolated using Triton X-100 and Brij-96 differ in their protein content.

Amino Acid Sequence↗
PubMed2026

Exploring the proteomic landscape of THP-1 monocytes through two-challenge LPS induction.

Proteome remodelling is central to the regulation of innate immune activation, yet the temporal organisation of protein networks engaged during repeated lipopolysaccharide (LPS) stimulation remains incompletely defined. In the present study, label-free quantitative mass spectrometry-based proteomics was used to characterise protein abundance changes in THP-1 monocytes at early (30 min) and later (2 h) time points following a second LPS challenge. This analysis was complemented by an independent co-immunoprecipitation proteomics experiment designed to identify candidate proteins associated with the regulatory pseudo-kinase IRAK3 during early TLR4 signalling. At 30 min, differentially abundant proteins were enriched in pathways associated with pattern-recognition receptor signalling, NF-κB activity, RNA processing, phosphorylation, and ribonucleoprotein complex organisation. By 2 h, the proteomic response broadened to include oxidative phosphorylation, antigen processing and presentation, vesicle-mediated transport, protein folding, and cytokine-regulatory pathways. These findings indicate that repeated LPS stimulation is accompanied by progressive remodelling of inflammatory, metabolic, translational, and proteostatic programmes rather than major changes in protein identity. Co-immunoprecipitation identified established TLR/IRAK3-associated components together with candidate IRAK3-associated proteins linked to RNA regulation, kinase signalling, ubiquitin-mediated processes, redox control, cytoskeletal remodelling, and damage-associated molecular pattern responses. Collectively, these findings define a temporal framework of proteomic adaptation during repeated inflammatory stimulation and expand the range of candidate proteins potentially contributing to IRAK3-centred regulation of innate immune signalling.

Humans↗
PubMed2006

Gel-free mass spectrometry-based high throughput proteomics: tools for studying biological response of proteins and proteomes.

Revolutionary advances in biological mass spectrometry (MS) have provided a basic tool to make possible comprehensive proteomic analysis. Traditionally, two-dimensional gel electrophoresis has been used as a separation method coupled with MS to facilitate analysis of complex protein mixtures. Despite the utility of this method, the many challenges of comprehensive proteomic analysis has motivated the development of gel-free MS-based strategies to obtain information not accessible using two-dimensional gel separations. These advanced strategies have enabled researchers to dig deeper into complex proteomes, gaining insights into the composition, quantitative response, covalent modifications and macromolecular interactions of proteins that collectively drive cellular function. This review describes the current state of gel-free, high throughput proteomic strategies using MS, including (i) the separation approaches commonly used for complex mixture analysis; (ii) strategies for large-scale quantitative analysis; (iii) analysis of post-translational modifications; and (iv) recent advances and future directions. The use of these strategies to make new discoveries at the proteome level into the effects of disease or other cellular perturbations is discussed in a variety of contexts, providing information on the potential of these tools in electromagnetic field research.

Animals↗
PubMedDate not supplied

Proteome analysis of low-abundance proteins using multidimensional chromatography and isotope-coded affinity tags.

The effectiveness of proteome-wide protein identification and quantitative expression profiling is dependent on the ability of the analytical methodologies employed to routinely obtain information on low-abundance proteins, as these are frequently of great biological importance. Two-dimensional gel electrophoresis, the traditional method for proteome analysis, has proven to be biased toward highly expressed proteins. Recently, two-dimensional chromatography of the complex peptide mixtures generated by the digestion of unseparated protein samples has been introduced for the identification of their components, and isotope-coded affinity tags (ICAT) have been introduced to allow for accurate quantification of the components of protein mixtures by mass spectrometry. Here, we demonstrate that the combination of isotope coded affinity protein tags and multidimensional chromatography/mass spectrometry of tryptic peptide mixtures is capable of detecting and quantifying proteins of low abundance in complex samples.

Animals↗
PubMed2002

Acid-labile isotope-coded extractants: a class of reagents for quantitative mass spectrometric analysis of complex protein mixtures.

Quantitative mass spectrometry using stable isotope-labeled tagging reagents such as isotope-coded affinity tags has emerged as a powerful tool for identification and relative quantitation of proteins in current proteomic studies. Here we describe an integrated approach using both automated two-dimensional liquid chromatography/ mass spectrometry (2D-LC/MS) and a novel class of chemically modified resins, termed acid-labile isotope-coded extractants (ALICE), for quantitative mass spectrometric analysis of protein mixtures. ALICE contains a thiol-reactive group that is used to capture all cysteine (Cys)-containing peptides from peptide mixtures, an acid-labile linker, and a nonbiological polymer. The acid-labile linker is synthesized in both heavy and light isotope-coded forms and therefore enables the direct relative quantitation of peptides/proteins through mass spectrometric analysis. To test the ALICE method for quantitative protein analysis, two model protein mixtures were fully reduced, alkylated, and digested in solution separately and then Cys-containing peptides covalently captured by either light or heavy ALICE. The reacted light and heavy ALICE were mixed and washed extensively under rigorous conditions and the Cys-containing peptides retrieved by mild acid-catalyzed elution. Finally, the eluted peptides were directly subjected to automated 2D-LC/MS for protein identification and LC/MS for accurate relative quantitation. Our initial study showed that quantitation of protein mixtures using ALICE was accurate. In addition, isolation of Cys-containing peptides by the ALICE method was robust and specific and thus yielded very low background in mass spectrometric studies. Overall, the use of ALICE provides improved dynamic range and sensitivity for quantitative mass spectrometric analysis of peptide or protein mixtures.

Amino Acid Sequence↗
PubMed2006

An ELISA-based procedure for assaying proteins in digests of human leukocytes and cell lines, using specifically selected peptides and appropriate antibodies.

BACKGROUND: We describe the application of an ELISA-based assay (the Peptidomatrix) that can be used to simultaneously identify and quantitate a number of proteins in biological samples. The biological sample (blood component, biopsy, culture or other) is first lysed to release all the proteins, without any additional separation. The denatured proteins in the sample are then digested in bulk with the desired proteolytic enzyme(s). The peptides in the digest are then assayed by appropriate antibodies, using a competition ELISA protocol. RESULTS: As an example of its use, the present paper applies the Peptidomatrix to the assay of four membrane proteins MDR1 (P-glycoprotein or ABCB1), MRP1 (ABCC1), BCRP/MXR (ABCG2) and the alpha subunit of the Na, K_ATPase (ATP1A1), present in a number of cell lines and in human lymphocytes. We show that we can detect and quantitate these proteins, using a series of peptide-antibody pairs, and that we can differentiate between cell lines or cell preparations that express the target proteins and those that do not. CONCLUSION: We have devised a simple, ELISA-based proteomics assay that enables the quantitation of designated proteins in a cell or tissue sample, and that can be used in any laboratory, with minimal specialized equipment.

Journal Article↗
PubMed2004

Proteomics in reproductive medicine: the technology for separation and identification of proteins.

With the near completion of the human genome project, reproductive biology is poised to enter the vastly more complex arena of proteomics. Proteomics involves the identification, characterization and quantitation of all proteins present in a cell at a particular metabolic state. Although the number of genes in the human genome is estimated to be about 27,000 +/- 5000, the number of proteins produced by humans is unknown, with estimates ranging as high as 1,500,000 distinct molecular entities. In order to address problems in the early stages of reproduction, proteomics must be scaled down to work with very few numbers of cells, termed zeptoproteomics. Mass spectrometry has rapidly become the key technology in proteomics, enabling rapid and facile identification and quantitation of femtomole and attomole quantities of a protein.

Chromatography, Affinity↗
PubMed2004

Quantitative profiling of LNCaP prostate cancer cells using isotope-coded affinity tags and mass spectrometry.

Androgens are involved in the pathogenesis of diseases including adenocarcinoma of the prostate. These hormones are important for growth, maintenance, and integrity of structure and function of the prostate. Androgen-deprivation is currently the only effective systemic therapy for prostate cancer but the effects of androgens on the proteome are still poorly described. Here we quantitatively profile changes in the proteome of LNCaP human prostate cancer cells in response to androgen using the newly developed isotope-coded affinity tag (ICAT) labeling and two-dimensional liquid chromatography-tandem mass spectroscopy (2-D LC-MS/MS). ICAT enables the concurrent identification and comparative quantitative analysis of proteins present in various biological samples including human cell and tissue extracts. Quantification and identification of 139 proteins in complex protein mixtures obtained from androgen-stimulated and unstimulated LNCaP cells were achieved. Changes in levels of 77 proteins in response to androgens were detected. Some of these proteins have been previously reported to be regulated by androgens and include spermine synthase, fatty acid synthase and calreticulin precursor. A large number of proteins that have not been previously reported to be expressed in prostate cells were also quantitatively identified. Examples of these include members of the dual specificity protein phosphatase subfamily, "similar" to hypothetical protein DKFZp434B0328.1, "similar" to 14-3-3 protein zeta and "similar" to hypothetical protein 458, components of the actin cytoskeleton and a range of unknown/uncharacterized proteins. This catalogue of proteins provides an overview of the proteome of prostate cancer cells and the global changes that occur in response to androgens.

Androgens↗
PubMed2005

Identification of fibrosis-relevant proteins using DIGE (difference in gel electrophoresis) in different models of hepatic fibrosis.

Proteomics became a more and more important technique for the large-scale analysis of proteins during the last years. Two-dimensional (2D) electrophoresis as a major tool of proteomics is a powerful method to compare two different biological stages (e. g. healthy and diseased tissue) and to find differences in their protein pattern. One major problem in proteomics is the gel to gel variation of two-dimensional gel electrophoresis, which could cause artefacts in the detection of expression differences. The "difference in gel electrophoresis" (DIGE) technique allows the separation of two proteomes in the same gel. The protein pools were labelled with different fluorescent dyes and equal amounts of protein were separated in the same gel. Another advantage of DIGE is the possibility to separate an internal standard labelled with a third dye in the same gel to allow quantitative expression analysis. We compared proteomes of three different fibrosis models with the appropriate control (tissue inhibitor of metalloproteinases-1 (TIMP-1) overexpressing HepG2 cells in comparison to a HepG2 control, freshly isolated HSC in comparison to activated HSC and healthy mouse liver in comparison to fibrotic mouse liver). Among the differentially expressed proteins several were already found to be relevant for fibrosis but we also detected some proteins like the selenium binding protein 2 which might be relevant for hepatic fibrosis.

Animals↗