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At least 919 records · Page 51Linked to original sources
PubMed2003

Protein identification: the origins of peptide mass fingerprinting.

Peptide mass fingerprinting (PMF) grew from a need for a faster, more efficient method to identify frequently observed proteins in electrophoresis gels. We describe the genesis of the idea in 1989, and show the first demonstration with fast atom bombardment mass spectrometry. Despite its promise, the method was seldom used until 1992, with the coming of significantly more sensitive commercial instrumentation based on MALDI-TOF-MS. We recount the evolution of the method and its dependence on a number of technical breakthroughs, both in mass spectrometry and in other areas. We show how it laid the foundation for high-throughput, high-sensitivity methods of protein analysis, now known as proteomics. We conclude with recommendations for further improvements, and speculation of the role of PMF in the future.

Amino Acid Sequence↗
PubMed2005

Sequence, annotation, and analysis of synteny between rice chromosome 3 and diverged grass species.

Rice (Oryza sativa L.) chromosome 3 is evolutionarily conserved across the cultivated cereals and shares large blocks of synteny with maize and sorghum, which diverged from rice more than 50 million years ago. To begin to completely understand this chromosome, we sequenced, finished, and annotated 36.1 Mb ( approximately 97%) from O. sativa subsp. japonica cv Nipponbare. Annotation features of the chromosome include 5915 genes, of which 913 are related to transposable elements. A putative function could be assigned to 3064 genes, with another 757 genes annotated as expressed, leaving 2094 that encode hypothetical proteins. Similarity searches against the proteome of Arabidopsis thaliana revealed putative homologs for 67% of the chromosome 3 proteins. Further searches of a nonredundant amino acid database, the Pfam domain database, plant Expressed Sequence Tags, and genomic assemblies from sorghum and maize revealed only 853 nontransposable element related proteins from chromosome 3 that lacked similarity to other known sequences. Interestingly, 426 of these have a paralog within the rice genome. A comparative physical map of the wild progenitor species, Oryza nivara, with japonica chromosome 3 revealed a high degree of sequence identity and synteny between these two species, which diverged approximately 10,000 years ago. Although no major rearrangements were detected, the deduced size of the O. nivara chromosome 3 was 21% smaller than that of japonica. Synteny between rice and other cereals using an integrated maize physical map and wheat genetic map was strikingly high, further supporting the use of rice and, in particular, chromosome 3, as a model for comparative studies among the cereals.

Arabidopsis↗
PubMed2026

Extracellular Vesicles From Glioblastoma Cells Reflect 2D vs. 3D Culture Adaptation and Resistance to Temozolomide.

Glioblastoma (GBM) is an aggressive brain tumor marked by extensive heterogeneity, resistance to therapy, and dismal prognosis. Extracellular vesicles (EVs) have emerged as key players in GBM biology, mediating intercellular communication and therapy adaptation. However, the exact functions and molecular impact of EVs in GBM remain incompletely understood. In this study, we performed a comparative proteomic analysis of U87MG GBM cells grown in two-dimensional (2D) monolayers and three-dimensional (3D) spheroids following temozolomide (TMZ) treatment, alongside characterization of EVs derived from both culture systems. 3D-spheroids secreted more EVs of smaller size and exhibited a more TMZ-resistant, stem-like proteome under TMZ-induced genotoxic stress. In contrast, 2D cell cultures demonstrated greater proteome remodeling, with EVs enriched in protein families involved in DNA repair, oxidative stress adaptation, and methylation processes. Notably, several methyltransferases were decreased intracellularly but selectively retained in EVs, suggesting active sorting to influence the tumor microenvironment or modulate epigenetic states in recipient cells. EVs also carried adhesion molecules and signaling proteins linked to migration, invasion, and Wnt pathway activation, as well as metabolic enzymes connecting serine metabolism and redox control to TMZ resistance. Mapping EV and cellular proteomes onto The Cancer Genome Atlas (TCGA) dataset identified prognostic protein families associated with either poor or favorable patient outcomes. Our data demonstrate that EV cargo composition mirrors TMZ-induced phenotypic adaptation and reveals molecular mechanisms underlying therapeutic resistance. These EV-associated signatures may serve as clinically actionable biomarkers for patient stratification and offer potential targets to overcome chemoresistance in GBM.

Humans↗
PubMed2005

The lipolytic proteome of mouse adipose tissue.

Hydrolysis of triacylglycerols and cholesteryl esters is a key event in energy homeostasis of animals. However, many lipolytic activities still await their molecular identification. Here we report on a novel tool for concomitant analysis of lipases in complex proteomes. Fluorescent activity tags mimicking lipid substrates were used to label the proteome of mouse adipose tissue. Analysis by two-dimensional gel electrophoresis and LC-MS/MS led to the identification of all known intracellular lipases as well as a number of novel candidates. One of them was recently shown to be involved in triacylglycerol mobilization in adipocytes and therefore named adipose triglyceride lipase. Functional characterization of expressed enzymes demonstrated that lipolytic and esterolytic activities could be well discriminated. Thus our results show the first map of the lipolytic proteome of mouse adipose tissue and demonstrate the general applicability of our method for rapid profiling and identification of lipolytic activities in complex biological samples.

4-Chloro-7-nitrobenzofurazan↗
PubMed2000

Profiling of Caenorhabditis elegans proteins using two-dimensional gel electrophoresis and matrix assisted laser desorption/ionization-time of flight-mass spectrometry.

The nematode Caenorhabditis elegans (C. elegans) is the first animal whose whole 97 Mb genome sequence, encoding ca. 19000 open reading frames (ORF's), has been essentially determined. We tried to establish a 2-DE map of the nematode proteome by means of two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). A soluble protein fraction of mixed stages of the worm, wild-type strain N2, was applied to 2-D PAGE. After Coomassie Brilliant Blue (CBB) staining, 1200 spots were detected and 140 major spots were excised from the gel and subjected to in-gel digestion with Achromobacter protease I (lysyl endopeptidase). Resulting peptides were analyzed by matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) followed by peptide mass fingerprinting for protein identification. With this approach we have obtained a two-dimensional electrophoresis (2-DE) protein map in which 69 spots were localized as landmarks for comparison of expression profiles to elucidate the basis of various biological events.

Animals↗
PubMed2001

New perspectives in the Escherichia coli proteome investigation.

Escherichia coli is a model organism for biochemical and biological studies as it is one of the best characterised prokaryote. Two-dimensional polyacrylamide gel electrophoresis, computer image analysis and different protein identification techniques gave rise, in 1995, to the Escherichia coli SWISS-2D PAGE database (http://www.expasy.ch/ch2d/). In the E. coli 3.5-10 SWISS-2D PAGE map, 40% of the E. coli proteome was displayed. The present study demonstrated that the use of narrow range pH gradients is able to potentially display up to a few copies of protein per E. coli cell. Moreover, the six new E. coli SWISS-2D PAGE maps (pH 4-5, 4.5-5.5, 5-6, 5.5-6.7, 6-9 and 6-11) presented here displayed altogether more than 70% of the entire E. coli proteome.

Databases, Protein↗
PubMed2005

Toward functional proteomics of alveolar macrophages.

Alveolar macrophages (AM) belong to a phenotype of macrophages with distinct biological functions and important pathophysiological roles in lung health and disease. The molecular details determining AM differentiation from blood monocytes and AM roles in lung homeostasis are largely unknown. With the use of different technological platforms, advances in the field of proteomics have made it possible to search for differences in protein expression between AM and their precursor monocytes. Proteome features of each cell type provide new clues into understanding mononuclear phagocyte biology. In-depth analyses using subproteomics and subcellular proteomics offer additional information by providing greater protein resolution and detection sensitivity. With the use of proteomic techniques, large-scale mapping of phosphorylation differences between the cell types have become possible. Furthermore, two-dimensional gel proteomics can detect germline protein variants and evaluate the impact of protein polymorphisms on an individual's susceptibility to disease. Finally, surface-enhanced laser desorption and ionization (SELDI) time-of-flight mass spectrometry offers an alternative method to recognizing differences in protein patterns between AM and monocytes or between AM under different pathological conditions. This review details the current status of this field and outlines future directions in functional proteomic analyses of AM and monocytes. Furthermore, this review presents viewpoints of integrating proteomics with translational topics in lung diseases to define the mechanisms of disease and to uncover new diagnostic and therapeutic targets.

Electrophoresis, Gel, Two-Dimensional↗
PubMed2005

Identification of differentially expressed proteins of gamma-ray irradiated rat intestinal epithelial IEC-6 cells by two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionisation-time of flight mass spectrometry.

To identify proteins involved in the processes of cellular and molecular response to radiation damage repair in intestinal epithelial IEC-6 cells, we comparatively analyzed the proteome of irradiated IEC-6 cells with that of normal cells. A series of methods were used, including two-dimensional gel electrophoresis (Z-DE), PDQuest software analysis of 2-DE gels, peptide mass fingerprinting based on matrix-assisted laser desorption/ionisation-time of flight-mass spectrometry (MALDI-TOF-MS), and Swiss-Prot database searching, to separate and identify differentially expressed proteins. Western blotting and reverse transcriptase polymerase chain reaction (RT-PCR) were used to validate the differentially expressed proteins. Image analysis revealed that averages of 608 +/- 39 and 595 +/- 31 protein spots were detected in normal and irradiated IEC-6 cells, respectively. Sixteen differential protein spots were isolated from gels, and measured with MALDI-TOF-MS. A total of 14 spots yielded good spectra, and 11 spots matched with known proteins after database searching. These proteins were mainly involved in anti-oxidation, metabolism, and protein post-translational processes. Western blotting confirmed that stress-70 protein was down-regulated by gamma-irradiation. Up-regulation of ERP29 was confirmed by RT-PCR, indicating that it is involved in ionizing radiation. The clues provided by the comparative proteome strategy utilized here will shed light on molecular mechanisms of radiation damage repair in intestinal epithelial cells.

Animals↗
PubMed2004

Identification of proteins in activated human neutrophils susceptible to tyrosyl radical attack. A proteomic study using a tyrosylating fluorophore.

Tyrosyl radicals cross-linked to protein tyrosine residues (tyrosylated proteins) represent hallmarks of neutrophil-mediated injury at the inflammatory locus. Yet the proteins targeted by tyrosyl radicals in an intact cellular system remain to be elucidated. Here, we show that tyrosyl radicals generated by human neutrophils after activation by phorbol 12-myristate 13-acetate (PMA), interferon-gamma (IFN-gamma) or TNF-alpha could act in an autocrine manner by cross-linking to endogenous proteins. We have identified the tyrosylated proteins by using a membrane-impermeable tyrosine analogue, tyramine coupled to fluorescein (TyrFluo), in combination with proteomics techniques. Confocal microscopy images indicated that initially the tyrosylated proteins were localized in patches at the cell surface to become internalized subsequently. In the neutrophil membrane-associated proteome, lactoferrin was the prime target of tyrosylation. Out of three isoforms identified, an 80 kDa neutral isoform was tyrosylated more extensively than the 85 kD basic isoform, particularly after PMA activation. Although all three stimuli induced tyrosylation of the filamentous component vimentin, additional tyrosylated vimentin fragments were detected after IFN-gamma- and TNF-alpha-stimulation. Moreover, upon activation the bulk of vimentin behaved as a dimer (M(r) 120 kDa) being slightly tyrosylated, yet phosphorylated at Thr-425 possibly as a requirement for its externalization. Unexpectedly, bovine catalase added to end tyrosyl radicals formation was detected as a highly tyrosylated neutrophil-associated protein. A moderate stimulus-dependent tyrosylation of ATP synthase-beta, alpha-enolase, glyceraldehyde 3-phosphate dehydrogenase, cytokeratin-10, filamin-A, and annexin-I was also observed. When the membrane-permeable probe (acetylTyrFluo) was used, protein tyrosylation was not observed indicating that the intracellular proteins were well protected against oxidative attack. This study shows that human neutrophils can modulate their proteome via a tyrosine oxidation pathway induced by pro-inflammatory mediators.

Animals↗
PubMed2003

Progress in the definition of a reference human mitochondrial proteome.

Owing to the complexity of higher eukaryotic cells, a complete proteome is likely to be very difficult to achieve. However, advantage can be taken of the cell compartmentalization to build organelle proteomes, which can moreover be viewed as specialized tools to study specifically the biology and "physiology" of the target organelle. Within this frame, we report here the construction of the human mitochondrial proteome, using placenta as the source tissue. Protein identification was carried out mainly by peptide mass fingerprinting. The optimization steps in two-dimensional electrophoresis needed for proteome research are discussed. However, the relative paucity of data concerning mitochondrial proteins is still the major limiting factor in building the corresponding proteome, which should be a useful tool for researchers working on human mitochondria and their deficiencies.

Databases as Topic↗
PubMed2004

FTICR mass spectrometry for qualitative and quantitative bioanalyses.

Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) is playing an increasing role in the characterization of cellular systems owing to its capabilities for providing higher confidence of identification, increased dynamic range and sensitivity unmatched by other MS platforms. Particularly in proteomics, where global and quantitative approaches are essential, the attributes of FTICR-MS are poised to make significant contributions. Recent advances in the field that have particular importance for proteomic applications include the use of high-performance micro-capillary column separation techniques coupled to FTICR, as well as methods that improve protein identification, sensitivity, dynamic range and throughput.

Animals↗
PubMed2001

Quantitative analysis of bacterial and mammalian proteomes using a combination of cysteine affinity tags and 15N-metabolic labeling.

We describe the combined use of 15N-metabolic labeling and a cysteine-reactive biotin affinity tag to isolate and quantitate cysteine-containing polypeptides (Cys-polypeptides) from Deinococcus radiodurans as well as from mouse B16 melanoma cells. D. radiodurans were cultured in both natural isotopic abundance and 15N-enriched media. Equal numbers of cells from both cultures were combined and the soluble proteins extracted. This mixture of isotopically distinct proteins was derivatized using a commercially available cysteine-reactive reagent that contains a biotin group. Following trypsin digestion, the resulting modified peptides were isolated using immobilized avidin. The mixture was analyzed by capillary reversed-phase liquid chromatography (LC) online with ion trap mass spectrometry (MS) as well as Fourier transform ion cyclotron resonance (FTICR) MS. The resulting spectra contain numerous pairs of Cyspolypeptides whose mass difference corresponds to the number of nitrogen atoms present in each of the peptides. Designation of Cys-polypeptide pairs is also facilitated by the distinctive isotopic distribution of the 15N-labeled peptides versus their 14N-labeled counterparts. Studies with mouse B16 cells maintained in culture allowed the observation of hundreds of isotopically distinct pairs of peptides by LC-FTICR analysis. The ratios of the areas of the pairs of isotopically distinct peptides showed the expected 1:1 labeling of the 14N and 15N versions of each peptide. An additional benefit from the present strategy is that the 15N-labeled peptides do not display significant isotope-dependent chromatographic shifts from their 14N-labeled counterparts, therefore improving the precision for quantitating peptide abundances. The methodology presented offers an alternate, cost-effective strategy for conducting global, quantitative proteomic measurements.

Animals↗
PubMed2006

Improved proteomic discovery by sample pre-fractionation using dual-column ion-exchange high performance liquid chromatography.

Clinically relevant biomarkers are urgently needed for improving patient diagnosis, risk stratification, prognosis and therapeutic treatments. There is a particularly compelling motivation for identifying protein-based indicators of early-stage disease for more effective interventions. Despite recent progress, the proteomic discovery process remains a daunting challenge due to the sheer heterogeneity and skewed protein abundances in biofluids. Even the most advanced mass spectrometry systems exhibit limiting overall dynamic ranges and sensitivities relative to the needs of modern biomedical applications. To this end, we report the development of a robust, rapid, and reproducible high performance ion-exchange liquid chromatography pre-fractionation method that allows for improved proteomic detection coverage of complex biological specimens using basic tandem mass spectrometry screening procedures. This form of sample simplification prior to global proteomic profiling, which we refer to collectively as 'fractionomics', increases the number and diversity of proteins that can be confidently identified in tissue and cell lysates as compared to the straight analysis of unfractionated crude extracts.

Animals↗
PubMed2005

Proteomics strategies for protein identification.

The information from genome sequencing provides new approaches for systems-wide understanding of protein networks and cellular function. DNA microarray technologies have advanced to the point where nearly complete monitoring of gene expression is feasible in several organisms. An equally important goal is to comprehensive survey cellular proteomes and profile protein changes under different cellular states. This presents a complex analytical problem, due to the chemical variability between proteins and peptides. Here, we discuss strategies to improve accuracy and sensitivity of peptide identification, distinguish represented protein isoforms, and quantify relative changes in protein abundance.

Computational Biology↗
PubMed2001

Proteomics as a tool in the pharmaceutical drug design process.

Proteomics is a technology platform that is gaining widespread use in drug discovery and drug development programs. Defined as the protein complement of the genome, the proteome is a varied and dynamic repertoire of molecules that in many ways dictates the functional form that is taken by the genome. The importance of proteomics is a direct consequence of the central role that proteins play in establishing the biological phenotype of organisms in healthy and diseased states. Moreover, proteins constitute the vast majority of drug targets against which pharmaceutical drug design processes are initiated. By studying interrelationships between proteins that occur in health and disease and following drug treatment, proteomics contributes important insight that can be used to determine the pathophysiological basis for disease and to study the mechanistic basis for drug action and toxicity. Proteomics is also an effective means to identify biomarkers that have the potential to improve decision making surrounding drug efficacy and safety issues based on data derived from the study of key tissues and the discovery and appropriate utilization of biomarkers.

Chromatography, High Pressure Liquid↗
PubMed2005

[Two-dimensional electrophoresis and mass spectrometry analysis on effects of trichloroethylene on protein of L-02 liver cells].

OBJECTIVE: To study the effects of trichloroethylene (TCE) on the protein in L-02 cells in vitro. METHODS: Thiazolyl blue and Trypan blue tests were used to investigate the cytotoxicity of TCE to L-02 liver cell. The 2-D electrophoresis was used to analyse the expression of proteins in L-02 liver cells. The differentially expressed protein spots were identified by matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-TOF-MS). RESULTS: When the concentration of TCE exceeded 30 micromol/L, there was distinct cytotoxicity to L-02 cell (P < 0.05). Selected 40 micromol/L to treat L-02 liver cells and analyze the differential proteome expression, the results showed that the expression level of 37 protein spots was up-regulated and 15 protein spots was down-regulated. And 15 proteins were identified by MALDI-TOF-TOF-MS. CONCLUSION: TCE can change the proteome expression of L-02 liver cell. It should provide the fundamental information to identify proteins related to TCE in further study.

Anesthetics, Inhalation↗
PubMed2005

High-resolution functional proteomics by active-site peptide profiling.

Characterization and functional annotation of the large number of proteins predicted from genome sequencing projects poses a major scientific challenge. Whereas several proteomics techniques have been developed to quantify the abundance of proteins, these methods provide little information regarding protein function. Here, we present a gel-free platform that permits ultrasensitive, quantitative, and high-resolution analyses of protein activities in proteomes, including highly problematic samples such as undiluted plasma. We demonstrate the value of this platform for the discovery of both disease-related enzyme activities and specific inhibitors that target these proteins.

Animals↗