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At least 73 records · Page 4Linked to original sources
PubMed2025

A proteomic map of thromboinflammatory signatures in antiphospholipid syndrome: results from antiphospholipid syndrome alliance for clinical trials and international networking (APS ACTION) registry.

INTRODUCTION: Antiphospholipid syndrome (APS) is an autoimmune disease with thromboembolic and obstetric morbidity arising via a model of immunothrombosis. Individuals with APS may present with thrombotic (TAPS), obstetric (OAPS), or microvascular (MAPS) disease, while many have circulating antiphospholipid antibodies (aPL) without APS classification (NoAPS). Multiple pathophysiologic mechanisms have been proposed in APS, including activation by aPL of platelets, endothelial and immune cells, as well as complement and coagulation pathways; however, the pathophysiology of APS, particularly transition of clinical APS from aPL remains unclear. METHODS: Seeking to define the inflammatory signature of APS, we carried out an unbiased proteomic screen of persistently aPL-positive patients with different clinical phenotypes from the international APS Alliance for Clinical Trials and International Networking (ACTION) Registry and compared them to 10 healthy controls. 6398 unique proteins were estimated using an DNA aptamer-based assay. Subsequently, we validated our findings in 34 additional patients. RESULTS: Our data show that the mere presence of aPL confers a distinct thromboinflammatory signature characterized by the activation of coagulation, complement, innate and adaptive immune response pathways shared by all APS subtypes. Pathway enrichment analysis revealed increasing enrichment with rising statistical significance of thrombosis, complement, neutrophil and other innate and adaptive immune activation, as well as extracellular matrix (ECM) organization with increasing clinical severity, suggesting a model of progressive thromboinflammation in evolution of APS from NoAPS to TAPS and MAPS. CONCLUSIONS: Our findings provide novel insights into the pathogenesis of APS and identify potential novel targets for diagnostic and therapeutic intervention in APS across its entire spectrum.

Humans↗
PubMed2026

Time- and Dose-Resolved DIA-PASEF Proteomics Maps the Transition from Adaptive Stress to Apoptotic Collapse in Melittin-Treated MDA-MB-231 Cells.

Melittin, the cytolytic peptide of honeybee venom, exhibits potent anticancer activity in triple-negative breast cancer (TNBC), yet the molecular programs underlying its cytotoxic effects remain incompletely defined. To address this gap, MDA-MB-231 TNBC cells were exposed to melittin at half-maximal inhibitory concentration(half IC50) and IC50 across early(0.5, 1, and 2 h), mid(3, 4 h), and late (12, 24 h) time windows. Proteomic profiling was performed using label-free data-independent acquisition(DIA) parallel accumulation-serial fragmentation(PASEF). Approximately 5800 proteins were quantified, revealing distinct dose-dependent stress responses. An integrative exploratory framework combining time-resolved log2 fold-change trajectories, area-under-the-curve(AUC) based temporal prioritization, and independent heatmap visualization identified proteins associated with melittin-induced stress remodeling. Half IC50 exposure showed a transient stress-adaptive signature characterized by chromatin remodeling(HMGN2, H2AZ1), structural and RNA-associated buffering(LRRC7), and indirect mitochondrial quality-control signaling(CPAMD8, SPATA4), which progressively weakened over time. In contrast, IC50 treatment induced rapid chromatin remodeling dominated by histone H1 variants(H1.4, H1.2), early RNA instability(LRRC7), and late-stage cytoskeletal disassembly marked by MICAL3 induction, consistent with progression toward apoptosis. These trajectories paralleled dose-dependent apoptotic phenotypes. Overall, data suggest that melittin elicits dose- and time-dependent proteomic stress responses in TNBC cells and identify candidate trajectory-associated proteins and pathways linked to adaptive stress remodeling or progression toward cytotoxic collapse.

Melitten↗
PubMed1998

Comprehensive two-dimensional high-performance liquid chromatography for the isolation of overexpressed proteins and proteome mapping.

A two-dimensional liquid chromatographic system is described here which uses size-exclusion liquid chromatography (SEC) followed by reversed-phase liquid chromatography (RPLC) to separate the mixture of proteins resulting from the lysis of Escherichia coli cells and to isolate the proteins that they produce. The size-exclusion chromatography can be conducted under either denaturing or nondenaturing conditions. Peaks eluting from the first dimension are automatically subjected to reversed-phase chromatography to separate similarly sized proteins on the basis of their various hydrophobicities. The RPLC also serves to desalt the analytes so that they can be detected in the deep ultraviolet region at 215 nm regardless of the SEC mobile phase used. The two-dimensional (2D) chromatograms produced in this manner then strongly resemble the format of stained 2D gels, in that spots are displayed on a X-Y axis and intensity represents quantity of analyte. Following chromatographic separation, the analytes are deposited into six 96-well (576 total) polypropylene microtiter plates via a fraction collector. Interesting fractions are analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF/MS) or electrospray mass spectrometry (ESI/MS) depending on sample concentration, which both yield accurate (2 to 0.02%) molecular weight information on intact proteins without any additional sample preparation, electroblotting, destaining, etc. The remaining 97% of a fraction can then be used for other analyses, such Edman sequencing, amino acid analysis, or proteolytic digestion and sequencing by tandem mass spectrometry. This 2D HPLC protein purification and identification system was used to isolate the src homology (SH2) domain of the nonreceptor tyrosine kinase pp60c-src and beta-lactamase, both inserted into E. coli, as well as a number of native proteins comprising a small portion of the E. coli proteome.

Bacterial Proteins↗
PubMed2006

Towards a lung adenocarcinoma proteome map: studies with SP-C/c-raf transgenic mice.

We report mapping of proteins of adenocarcinomas of the lung as a result of overexpression of the oncogenically activated N-terminal deletion mutant c-raf-1 BxB through usage of the human SP-C promotor. Proteins from non-transgenic controls and tumors were extracted with a lysis buffer containing 5 mol/L urea, 2 mol/L thiourea, 40 mmol/L Tris, 4% CHAPS, 100 mmol/L DTT, 0.5% BioLyte 3-10, separated by 2-DE and studied by image analysis. On average, 300-600 protein spots per gel were excised and analyzed by MALDI-TOF and -TOF/TOF MS. More than 1000 of the CBB-stained proteins were identified and traced back to 100 different gene products, including many of their isoforms. We observed significant changes in the expression of proteins involved in cellular defense or glycolysis, and this included glutathione S-transferase, peroxiredoxin 6, and alpha-enolase, among others. Proteins associated with lung tumor growth and/or metastasis, i.e., lung carbonyl reductase, differed in expression, as did tumor-associated expression of cell adhesion and membrane-bound proteins such as vinculin. This map provides valuable insight into expression of pulmonary proteins associated with lung adenocarcinomas, some of which may be of utility as diagnostic markers in clinical trials.

Adenocarcinoma↗
PubMed2006

Proteomic mapping of a suppressor of non-chemotactic cheW mutants reveals that Helicobacter pylori contains a new chemotaxis protein.

Bacterial chemotaxis is a colonization factor for the ulcer-causing pathogen Helicobacter pylori. H. pylori contains genes encoding the chemotaxis signalling proteins CheW, CheA and CheY; CheW couples chemoreceptors to the CheA kinase and is essential for chemotaxis. While characterizing a cheW mutant, we isolated a spontaneous, chemotactic variant (Che+). We determined that this phenotype was caused by a genetic change unlinked to the original cheW mutation. To locate the underlying Che+ mutation, we compared total protein profiles of the non-chemotactic mutant (cheW) with those from the cheW Che+ variant by two-dimensional differential in-gel electrophoresis. One protein was found only in the cheW Che+ variant. This protein was identified by MS/MS as HP0170, a hypothetical protein with no known function. DNA sequencing verified that hp0170 was mutated in the cheW Che+ suppressor, and deletion of this open reading frame in the cheW background nearly recapitulated the Che+ suppressor phenotype. Using hidden Markov models, we found that HP0170 is a remote homologue of E. coli CheZ. CheZ interacts with phosphorylated CheY and stimulates its autodephosphorylation. CheZ was not predicted to be present in epsilon-proteobacteria. We found that chemotaxis in the cheW Che+ suppressor depended on both cheY and cheA. We hypothesize that a small amount of phosphorylated CheY is generated via CheA in the cheW mutant, and this amount is sufficient to affect flagellar rotation when HP0170 is removed. Our results suggest that HP0170 is a remote homologue of CheZ, and that CheZ homologues are found in a broader range of bacteria than previously supposed.

Amino Acid Sequence↗
PubMedDate not supplied

Sensitivity and mass accuracy for proteins analyzed directly from polyacrylamide gels: implications for proteome mapping.

Matrix-assisted laser desorption ionization (MALDI) mass spectra have been obtained directly from thin-layer isoelectric focusing (IEF) gels with as little as 700 femtomoles of alpha- and beta-chain bovine hemoglobin and bovine carbonic anhydrase, and 2 picomoles of bovine trypsinogen, soybean trypsin inhibitor, and bovine serum albumin all loaded onto a single lane. By soaking the gel in a matrix solution, matrix was deposited over the entire gel surface, allowing MALDI scanning down complete lanes of the one-dimensional gel. As long as matrix crystals were deposited finely on the surface of the gel, time-lag focusing techniques were capable of ameliorating some of the mass accuracy limitations inherent in desorbing from uneven insulator surfaces with external calibration. Eleven measurements on the 5 kDa alpha-subunit proteins of lentil lectin measured over the course of 1 h and referenced to a single calibration yielded a standard deviation of 0.025%. Colloidal gold staining was found to be compatible with desorption directly from IEF and sodium dodecyl sulfate (SDS)-polyacrylamide gels. This direct approach simplifies the interface between gel electrophoresis and mass spectrometry dramatically, making the process more amenable to automation.

Acrylic Resins↗
PubMed2003

Mapping the proteome of Leishmania Viannia parasites using two-dimensional polyacrylamide gel electrophoresis and associated technologies.

In this study we have demonstrated the potential of two-dimensional electrophoresis (2DE)-based technologies as tools for characterization of the Leishmania proteome (the expressed protein complement of the genome). Standardized neutral range (pH 5-7) proteome maps of Leishmania (Viannia) guyanensis and Leishmania (Viannia) panamensis promastigotes were reproducibly generated by 2DE of soluble parasite extracts, which were prepared using lysis buffer containing urea and nonidet P-40 detergent. The Coomassie blue and silver nitrate staining systems both yielded good resolution and representation of protein spots, enabling the detection of approximately 800 and 1,500 distinct proteins, respectively. Several reference protein spots common to the proteomes of all parasite species/strains studied were isolated and identified by peptide mass spectrometry (LC-ES-MS/MS), and bioinformatics approaches as members of the heat shock protein family, ribosomal protein S12, kinetoplast membrane protein 11 and a hypothetical Leishmania-specific 13 kDa protein of unknown function. Immunoblotting of Leishmania protein maps using a monoclonal antibody resulted in the specific detection of the 81.4 kDa and 77.5 kDa subunits of paraflagellar rod proteins 1 and 2, respectively. Moreover, differences in protein expression profiles between distinct parasite clones were reproducibly detected through comparative proteome analyses of paired maps using image analysis software. These data illustrate the resolving power of 2DE-based proteome analysis. The production and basic characterization of good quality Leishmania proteome maps provides an essential first step towards comparative protein expression studies aimed at identifying the molecular determinants of parasite drug resistance and virulence, as well as discovering new drug and vaccine targets.

Animals↗
PubMedDate not supplied

Proteomic maps-toxicity relationship of halocarbons studied with similarity index and genetic algorithm.

In this work we analyzed proteomic maps obtained from hepatocytes, which were treated with 14 halocarbons. A similarity index was introduced as a robust measure of similarity between two maps or between two selections of spots within the maps. A searching algorithm was used to identify the spots that may play an important role in toxicity mechanism. The highest correlation coefficients obtained between the similarity index and biological parameter were larger than 0.9.

Algorithms↗
PubMed2006

Expanding the protein catalogue in the proteome reference map of human breast cancer cells.

In this report we present a catalogue of 162 proteins (including isoforms and variants) identified in a prototype of proteomic map of breast cancer cells. This work represents the prosecution of previous studies describing the protein complement of breast cancer cells of the line 8701-BC, which has been well characterized for several parameters, providing to be a useful model for the study of breast cancer-associated candidate biomarkers. In particular, 110 spots were identified ex novo by PMF, or validated following previous gel matching identification method; 30 were identified by N-terminal microsequencing and the remaining by gel matching with maps available from our former work. As a consequence of the expanded number of proteins, we have updated our previous classification extending the number of protein groups from 4 to 13. In order to facilitate comparative proteome studies of different kinds of breast cancers, in this report we provide the whole complement of proteins so far identified and grouped into the new classification. A consistent number of them were not described before in other proteomic maps of breast cancer cells or tissues, and therefore they represent a valuable contribution for breast cancer protein databases and for future application in basic and clinical researches.

Biomarkers, Tumor↗
PubMed2004

Proteome reference maps of vegetative tissues in pea. An investigation of nitrogen mobilization from leaves during seed filling.

A proteomic approach was used to analyze protein changes during nitrogen mobilization (N mobilization) from leaves to filling seeds in pea (Pisum sativum). First, proteome reference maps were established for mature leaves and stems. They displayed around 190 Coomassie Blue-stained spots with pIs from 4 to 7. A total of 130 spots were identified by mass spectrometry as corresponding to 80 different proteins implicated in a variety of cellular functions. Although the leaf proteome map contained more abundant spots, corresponding to proteins involved in energy/carbon metabolism, than the stem map, their comparison revealed a highly similar protein profile. Second, the leaf proteome map was used to analyze quantitative variations in leaf proteins during N mobilization. Forty percent of the spots showed significant changes in their relative abundance in the total protein extract. The results confirmed the importance of Rubisco as a source of mobilizable nitrogen, and suggested that in pea leaves the rate of degradation of Rubisco may vary throughout N mobilization. Correlated with the loss of Rubisco was an increase in relative abundance of chloroplastic protease regulatory subunits. Concomitantly, the relative abundance of some proteins related to the photosynthetic apparatus (Rubisco activase, Rubisco-binding proteins) and of several chaperones increased. A role for these proteins in the maintenance of a Rubisco activation state and in the PSII repair during the intense proteolytic activity within the chloroplasts was proposed. Finally, two 14-3-3-like proteins, with a potential regulatory role, displayed differential expression patterns during the massive remobilization of nitrogen.

Amino Acid Sequence↗
PubMed2003

Proteome analysis of male gametophyte development in rice anthers.

We used proteomic analysis to investigate the changing patterns of protein synthesis during pollen development in anthers from rice plants grown under strictly controlled growth conditions. Cytological analysis and external growth measurements such as anther length, auricle distances and days before flowering were used to determine pollen developmental stages. This allowed the collection of synchronous anther materials representing six discrete pollen developmental stages. Proteins were extracted from the anther samples and separated by two-dimensional gel electrophoresis to produce proteome maps. The anther proteome maps of different developmental stages were compared and 150 protein spots, which were changed consistently during development, were analysed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry to produce peptide mass fingerprint (PMF) data. Database searches using these PMF data revealed the identities of 40 of the protein spots analyzed. These 40 proteins represent 33 unique gene products. Four protein spots that could not be identified by PMF analysis were analysed by N-terminal microsequencing. Multiple charge-isoforms of vacuolar acid invertase, fructokinase, beta-expansin and profilin were identified. These proteins are closely associated with sugar metabolism, cell elongation and cell expansion, all of which are cell activities that are essential to pollen germination. The existence of multiple isoforms of the same proteins suggests that during the process of pollen development some kind of post-translational modification of these proteins occurs.

Amino Acid Sequence↗
PubMed2004

Towards two-dimensional electrophoresis mapping of the cerebrospinal fluid proteome from a single individual.

We test the ability of state-of-the-art two-dimensional electrophoresis (2-DE) technology to enable the proteome mapping of ante mortem cerebrospinal fluid (CSF) from a single individual. Using the sensitive technologies of a fluorescent protein stain and fluorescence laser scanning of 2-DE gels, combined with matrix-assisted laser desorption/ionization-time of flight/time of flight-mass spectrometry (MALDI-TOF/TOF-MS) for protein identification, a highly detailed 2-DE map of the CSF proteome was created. The 2-DE map contains 600 identified spots representing 82 different proteins. Of the 82 proteins identified, 25 have not appeared in any previously published 2-DE map of CSF, and 11 have not been previously reported to exist in CSF. Most of the identifications originate from an ante mortem CSF sample collected from a single hydrocephalus patient. A supplemental map created from neurologically normal patients is also presented. A webpage with protein identification and scoring information from both maps is available at http://www.leelab.org/csfmap.

Cerebrospinal Fluid↗
PubMed2004

A proteome reference map for Vibrio cholerae El Tor.

A proteome reference map has been constructed for Vibrio cholerae El Tor, in the pI range of 4.0 to 7.0. The map is based on two-dimensional gels (2-D) and the identification, by peptide mass fingerprint, of proteins in 94 spots, corresponding to 80 abundant proteins. Two strains are compared, strain N16961 and a Latin American El Tor strain C3294. The consensus map contains 340 spots consistently seen with both strains grown in Luria-Bertani broth (LB) or minimal M9 medium. The results were obtained from nine gels run with 18 cm immobilized pH gradient strips and precast gels. The 2-D gels were anchored to real N16961 proteins identified by mass spectrometry. Various energy metabolism components and periplasmic ATP-binding cassette (ABC) transporter proteins were identified among the abundant proteins. Two isoforms of OmpU were found. Five operons are proposed and seven hypothetical proteins were experimentally confirmed. Comparisons are made with protein 2-D gels for a classical strain and to microarray analysis available for the N16961 El Tor strain. New results were obtained from the proteome analysis, indicating an abundance of periplasmic ABC transporter proteins not found in microarray studies.

Bacterial Proteins↗
PubMed2006

Proteomic trajectory mapping of biological transformation: Application to developmental mouse retina.

In this report we introduce a new concept "proteomic trajectory mapping" for the investigation of a complex phenomenon underlying biological transformation and transition. We define proteomic trajectory to be the kinetic trace of protein expression and present a successful proteomic trajectory mapping of complex molecular events underlying postnatal development of mouse retina. Cluster analysis of the trajectory data using a two-state model identified four proteomic trajectory types: two distinct trajectory types accounting for the decline or the rise of protein molecules actively expressed in the juvenile stage (J-type) or in the adult stage (A-type), a class of transient trajectories that mediate the transformation from the juvenile to the adult stage (T-type), and the steady trajectories throughout the entire process of transformation (C-type). The dominance of particular protein categories expressed in each trajectory characterizes the stage of retinal development. Proteomic trajectory mapping will be a powerful tool to study the systematic changes of protein expression caused by physiological, genetic, or pathological agents and the reverse of such changes to the norm by a treatment. The proteomic trajectory mapping is applicable to any biological transformation and, therefore, will be a powerful tool in biomedical sciences.

Animals↗
PubMed2006

Construction of quantitative proteome reference maps of mouse spleen and lymph node based on two-dimensional gel electrophoresis.

Quantitative features of the proteome are extremely useful for studying cellular processes at a molecular level. In this study, we attempted to construct quantitative reference proteome maps of the mouse spleen and lymph node based on 2-DE followed by protein identification using MS. We analyzed more than 1000 spots on the 2-DE images and consequently were able to determine that 919 spots were derived from 328 different genes. To obtain statistically reliable information of the protein levels from these 2-DE images, we measured the volumes of the respective spots on 2-DE images obtained by four to six independent experimental runs. These measurements were used to calculate the variability of the volumes of the respective spots on 2-DE following subcellular fractionation, which enabled us to discriminate differentially produced proteins from those within the range of intrinsic variability. More importantly, while the 2-DE data have been traditionally collected in a gel image-based manner, the resultant quantitative 2-DE data could be analyzed using the same procedure as that for mRNA expression profiles. This greatly assists in bridging the gap between the analyses of transcriptomes and proteomes and enables the integration of this data on the same informational platform.

Amino Acid Sequence↗
PubMed2001

Establishment of a root proteome reference map for the model legume Medicago truncatula using the expressed sequence tag database for peptide mass fingerprinting.

We have established a proteome reference map for Medicago truncatula root proteins using two-dimensional gel electrophoresis combined with peptide mass fingerprinting to aid the dissection of nodulation and root developmental pathways by proteome analysis. M. truncatula has been chosen as a model legume for the study of nodulation-related genes and proteins. Over 2,500 root proteins could be displayed reproducibly across an isoelectric focussing range of 4-7. We analysed 485 proteins by peptide mass fingerprinting, and 179 of those were identified by matching against the current M. truncatula expressed sequence tag (EST) database containing DNA sequences of approximately 105,000 ESTs. Matching the EST sequences to available plant DNA sequences by BLAST searches enabled us to predict protein function. The use of the EST database for peptide identification is discussed. The majority of identified proteins were metabolic enzymes and stress response proteins, and 44% of proteins occurred as isoforms, a result that could not have been predicted from sequencing data alone. We identified two nodulins in uninoculated root tissue, supporting evidence for a role of nodulins in normal plant development. This proteome map will be updated continuously (http://semele.anu.edu.au/2d/2d.html) and will be a powerful tool for investigating the molecular mechanisms of root symbioses in legumes.

Databases as Topic↗
PubMed2004

Proteome reference maps of Medicago truncatula embryogenic cell cultures generated from single protoplasts.

Using a combination of two-dimensional gel electrophoresis (2-DE) protein mapping and mass spectrometry (MS) analysis, we have established proteome reference maps of Medicago truncatula embryogenic tissue culture cells. The cultures were generated from single protoplasts, which provided a relatively homogeneous cell population. We used these to analyze protein expression at the globular stages of somatic embryogenesis, which is the earliest morphogenetic embryonic stage. Over 3000 proteins could reproducibly be resolved over a pI range of 4-11. Three hundred and twelve protein spots were extracted from colloidal Coomassie Blue-stained 2-DE gels and analyzed by matrix-assisted laser desorption/ionization-time of flight MS analysis and tandem MS sequencing. This enabled the identification of 169 protein spots representing 128 unique gene products using a publicly available expressed sequence tag database and the MASCOT search engine. These reference maps will be valuable for the investigation of the molecular events which occur during somatic embryogenesis in M. truncatula. The proteome reference maps and supplementary materials will be available and updated for public access at http://semele.anu.edu.au/.

Amino Acid Sequence↗
PubMed2002

Evaluation of proteome reference maps for cross-species identification of proteins by peptide mass fingerprinting.

We tested whether proteome reference maps established for one species can be used for cross-species protein identification by comparing two-dimensional protein gel patterns and protein identification data of two closely related bacterial strains and four plant species. First, proteome profiles of two strains of the fully sequenced bacterium Sinorhizobium meliloti were compared as an example of close relatedness, high reproducibility and sequence availability. Secondly, the proteome profiles of three legumes (Medicago truncatula, Melilotus alba and Trifolium subterraneum), and the nonlegume rice (Oryza sativa) were analysed to test cross-species similarities. In general, we found stronger similarities in gel patterns of the arrayed proteins between the two bacterial strains and between the plant species than could be expected from the sequence similarities. However, protein identity could not be concluded from their gel position, not even when comparing strains of the same species. Surprisingly, in the bacterial strains peptide mass fingerprinting was more reliable for species-specific protein identification than N-terminal sequencing. While peptide masses were found to be unreliable for cross-species protein identification, we present useful criteria to determine confident matching against species-specific expressed sequence tag databases. In conclusion, we present evidence that cautions the use of proteome reference maps and peptide mass fingerprinting for cross-species protein identification.

Electrophoresis, Gel, Two-Dimensional↗