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Results for “differential proteomics analysis”

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PubMed2005

Numerical approaches for quantitative analysis of two-dimensional maps: a review of commercial software and home-made systems.

The present review attempts to cover a number of methods that have appeared in the last few years for performing quantitative proteome analysis. However, due to the large number of methods described for both electrophoretic and chromatographic approaches, we have limited this review to conventional two-dimensional (2-D) map analysis which couples orthogonally a charge-based step (isoelectric focusing) to a size-based separation step (sodium dodecyl sulfate-electrophoresis). The first and oldest method applied to 2-D map data reduction is based on statistical analysis performed on sets of gels via powerful software packages, such as Melanie, PDQuest, Z3 and Z4000, Phoretix and Progenesis. This method calls for separately running a number of replicas for control and treated samples. The two sets of data are then merged and compared via a number of software packages which we describe. In addition to commercially-available systems, a number of home made approaches for 2-D map comparison have been recently described and are also reviewed. They are based on fuzzyfication of the digitized 2-D gel image coupled to linear discriminant analysis, three-way principal component analysis or a combination of principal component analysis and soft-independent modeling of class analogy. These statistical tools appear to perform well in differential proteomic studies.

Cluster Analysis↗

PubMed2003

Rediscovering the sweet spot in drug discovery.

Advances over the past decade in drug discovery technologies have not yet led to an increase in productivity. We analyzed the reasons that have led to this juncture and identify the selection of the right target and the right lead as crucial. New approaches are required to take full advantage of the genomics revolution. For targets, methods are becoming available for high-throughput proteome analysis and pathway characterization that synergize with studies of disease association and differential expression. For leads, methods are being developed that 'reverse' the high-throughput screening paradigm by mapping drugs and drug-like compounds back onto the proteome. The synergy between pathway mapping and compound mapping could allow the pharmaceutical and biotechnology industries to rediscover the sweet spot of research productivity.

Biotechnology↗

PubMed2005

Two-dimensional fluorescence difference gel electrophoresis analysis of the urine proteome in human diabetic nephropathy.

Urinary proteins may provide clues regarding pathogenesis of kidney disease as well as providing markers of disease activity. We employed two-dimensional differential in-gel electrophoretic analysis (2-D DIGE) to assess multiple urine samples in patients with diabetic nephropathy. Patient samples were collected as timed overnight collections. All the patients had longstanding diabetes, impaired renal function, and overt proteinuria. Control and patient urinary protein were analyzed by 2-D DIGE and DeCyder analysis. Ninety-nine spots were significantly regulated in the urine proteome of the diabetic samples, with 63 up- and 36 down-regulated. One spot corresponding to a pI 5-6 and a molecular weight between 45 and 66 kDa was consistently up-regulated by 19-fold across individuals in the diabetic group. Surface-enhanced laser desorption/ionization-time of flight analysis of in-gel tryptic digest of this spot identified this protein as alpha 1 antitrypsin (AAT). ELISA of urine samples from a separate group of patients and controls confirmed a marked increase of AAT in diabetic patients. Immunostaining of human diabetic kidneys revealed up-regulation of AAT in areas of renal fibrosis. In conclusion, we developed a method to analyze numerous urine samples from patients and allowed for detection and identification of regulated urine protein spots.

Aged↗

PubMed2025

Diversity and distribution of the lanthanome in aerobic methane-oxidising bacteria.

BACKGROUND: Lanthanides (Ln) play important and often regulatory roles in the metabolism of methylotrophs, including methanotrophs, particularly through their involvement in methanol oxidation. However, the diversity, distribution, and ecological relevance of Ln-associated proteins (the lanthanome) in aerobic methane-oxidising bacteria (MOB) remain underexplored. This study investigates the lanthanome using genome, plasmid, and proteome data, alongside metatranscriptome data from methane-rich lake sediments. RESULTS: We surveyed 179 genomes spanning Proteobacterial, Verrucomicrobial, and Actinobacterial MOBs to examine the distribution of Ln-dependent methanol dehydrogenases (MDHs) and Ln transport proteins. Distinct lineage-specific patterns were observed: XoxF5 was the most widespread MDH variant in Proteobacteria, while XoxF2 was restricted to Verrucomicrobia. Transporter systems also showed distinct patterns, with LanM restricted to Alphaproteobacteria, LanPepSY and LanA confined to Gammaproteobacteria, and LutH-like receptors broadly distributed across all lineages. Homologues of these genes were also detected on plasmids, indicating potential for horizontal gene transfer. In Lake Washington sediment metatranscriptomes, lanthanome transcripts were detected, with Proteobacteria as dominant contributors. Notably, a large fraction of xoxF transcripts were affiliated with non-MOB Methylophilaceae, consistent with known cooperative interactions with MOB. Using Methylosinus trichosporium OB3b as a model, we assessed methane oxidation and proteomic responses to soluble CeCl3 and a mixed-lanthanide ore. Lag phases were prolonged in the presence of lanthanides, particularly with ore, but methane oxidation rates converged across treatments after acclimation. Proteomic analysis revealed extensive condition-specific responses, with 724 proteins differentially expressed in Ore treatment compared to 60 under CeCl3. XoxF3 and XoxF5 were upregulated while MxaF and its accessory proteins were downregulated, consistent with the "lanthanide switch". Notably, LanM was not expressed despite being encoded, whereas LutH-like receptor was downregulated under both treatments, likely reflecting regulatory control to prevent excess metal uptake. Additional upregulation of a TonB-dependent receptor and ABC transporter suggests a potential lanthanophore-mediated uptake strategy. CONCLUSION: This study highlights the diversity and ecological activity of Ln-binding and transport systems in MOBs, their plasmid localisation and potential mobility, and their distinct regulation under different Ln sources. The strong proteomic response to complex ore underscores the physiological flexibility of MOBs in coping with natural lanthanide forms. These findings provide a framework for ecological studies and candidate targets for biotechnological applications in methane bioconversion and sustainable lanthanide recovery from complex materials.

Horizontal gene transfer↗

PubMed2006

Proteome and transcriptome analysis of 5-fluoro-2'-deoxyuridine-induced cell death mechanisms.

5-Fluoro-2'-deoxyuridine (FUdR) inhibits thymidylate synthase. The inhibition of thymidylate synthase causes an imbalance of intracellular deoxyribonucleoside triphosphate (dNTP) pools which subsequently induced cell death. We have been investigated the molecular mechanisms of cell death in mouse mammary tumor FM3A cells, F28-7 strain and its mutant F28-7-A strain, after treated with FUdR. We have previously been reported that F28-7 strain induced DNA cleavage into chromosomal sized fragments and subsequently develop necrosis, but F28-7-A strain induced DNA cleavage into oligonucleosomal sized fragments and subsequently develop apoptosis after treated with FUdR. In this report, in order to understand the molecular mechanisms of regulate of two differential cell death necrosis and apoptosis, we identify cell death regulator by using proteome and transcriptome analysis. When compared with the proteome from F28-7 strain and F28-7-A strain, it was found that ten proteins were increased and six proteins were decreased in F28-7-A strain. Furthermore, transcriptome analysis shows that 127 genes were increased and 181 genes were decreased in F28-7-A strain. These differentially expressed proteins and genes were involved in various cellular processes such as cell cycle regulation, apoptosis, proliferation, and differentiation. These two techniques clarified numerous features in F28-7 strain and F28-7-A strain. Our results revealed that numerous features indicated the coordinated regulation of molecular networks from various aspects of necrosis or apoptosis at the proteome and transcriptome levels.

Animals↗

PubMed2006

Proteomic analysis of nipple aspirate fluid from women with early-stage breast cancer using isotope-coded affinity tags and tandem mass spectrometry reveals differential expression of vitamin D binding protein.

BACKGROUND: Isotope-coded affinity tag (ICAT) tandem mass spectrometry (MS) allows for qualitative and quantitative analysis of paired protein samples. We sought to determine whether ICAT technology could quantify and identify differential expression of tumor-specific proteins in nipple aspirate fluid (NAF) from the tumor-bearing and contralateral disease-free breasts of patients with unilateral early-stage breast cancer. METHODS: Paired NAF samples from 18 women with stage I or II unilateral invasive breast carcinoma and 4 healthy volunteers were analyzed using ICAT labeling, sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE), liquid chromatography, and MS. Proteins were identified by sequence database analysis. Western blot analysis of NAF from an independent sample set from 12 women (8 with early-stage breast cancer and 4 healthy volunteers) was also performed. RESULTS: 353 peptides were identified from tandem mass spectra and matched to peptide sequences in the National Center for Biotechnology Information database. Equal numbers of peptides were up- versus down-regulated. Alpha2HS-glycoprotein [Heavy:Light (H:L) ratio 0.63] was underexpressed in NAF from tumor-bearing breasts, while lipophilin B (H:L ratio 1.42), beta-globin (H:L ratio 1.98), hemopexin (H:L ratio 1.73), and vitamin D-binding protein precursor (H:L ratio 1.82) were overexpressed. Western blot analysis of pooled samples of NAF from healthy volunteers versus NAF from women with breast cancer confirmed the overexpression of vitamin D-binding protein in tumor-bearing breasts. CONCLUSION: ICAT tandem MS was able to identify and quantify differences in specific protein expression between NAF samples from tumor-bearing and disease-free breasts. Proteomic screening techniques using ICAT and NAF may be used to find markers for diagnosis of breast cancer.

Biomarkers, Tumor↗

PubMed2005

DEPD: a novel database for differentially expressed proteins.

SUMMARY: The Differentially Expressed Protein Database was designed to store the output of comparative proteomics studies and provides a publicly available query and analysis platform for data mining. The database contains information about more than 3000 differentially expressed proteins (DEPs) manually extracted from the published literature, including relevant biological, experimental and methodological elements. Tools for visualization and functional analysis of DEPs are provided via a user-friendly webinterface. AVAILABILITY: http://protchem.hunnu.edu.cn/depd/.

Computational Biology↗

PubMed2005

Analysis of differential protein expression in Acidithiobacillus ferrooxidans grown under different energy resources respectively using SELDI-ProteinChip technologies.

Surface-enhanced laser desorption/ionization (SELDI)-time of flight is an affinity-based mass spectrometric method in which proteins of interest are selectively absorbed to a chemically modified surface on a chip, which allows proteomic analysis with limited material requirements. This characteristic makes it a valuable technique for microbiologists handling problematic samples, such as low cell number cultures. In this study, we explored differential-expressed proteome of Acidithiobacillus ferrooxidans cultivated with Fe(2+) and elemental sulfur separately by adopting the protein biochip SELDI approach. The cell lysates of A. ferrooxidans were applied onto Ciphergen ProteinChip WCX2, SAX2 and IMAC-Cu arrays. Proteins bound to the chips were analyzed on a ProteinChip Reader Model PBS II. A summary of the molecular masses of the differentially regulated proteins found on WCX2, IMAC-Cu and SAX2 was obtained and 28 differentially expressed proteins were found on the molecular weight range of 5.0 to 25 kDa.

Acidithiobacillus↗

PubMed2004

Proteome analysis of mouse macrophages treated with anthrax lethal toxin.

Anthrax toxin produced by Bacillus anthracis is a tripartite toxin comprising of protective antigen (PA), lethal factor (LF) and edema factor (EF). PA is the receptor-binding component, which facilitates the entry of LF or EF into the cytosol. EF is a calmodulin-dependent adenylate cyclase that causes edema whereas LF is a zinc metalloprotease and leads to necrosis of macrophages. It is also important to note that the exact mechanism of LF action is still unclear. With this view in mind, in the present study, we investigated a proteome wide effect of anthrax lethal toxin (LT) on mouse macrophage cells (J774A.1). Proteome analysis of LT-treated and control macrophages revealed 41 differentially expressed protein spots, among which phosphoglycerate kinase I, enolase I, ATP synthase (beta subunit), tubulin beta2, gamma-actin, Hsp70, 14-3-3 zeta protein and tyrosine/tryptophan-3-monooxygenase were found to be down-regulated, while T-complex protein-1, vimentin, ERp29 and GRP78 were found to be up-regulated in the LT-treated macrophages. Analysis of up- and down-regulated proteins revealed that primarily the stress response and energy generation proteins play an important role in the LT-mediated macrophage cell death.

Adenosine Triphosphate↗

PubMed2006

Protein expression profiling identifies maspin and stathmin as potential biomarkers of adenoid cystic carcinoma of the salivary glands.

Adenoid cystic carcinoma (ACC) is one of the most common malignant tumors of the salivary glands. It tends to grow slowly but is associated with a poor prognosis compared to other malignant salivary gland tumors. To identify specific markers of ACC, we examined protein expression profiling in ACC xenograft and normal salivary glands (NSG) using fluorescent 2-dimensional differential in-gel electrophoresis (2-D-DIGE), an emerging technique for comparative proteomics, that improves the reproducibility and reliability of differential protein expression analysis between the samples. To identify the proteins, matrix-assisted laser desorption/ionization time-of-flight peptide mass fingerprinting was carried out. Using these strategies, we detected 4 upregulated proteins and 5 downregulated proteins in ACC xenograft. Maspin and stathmin were selected for further analyses. Western blotting and immunohistochemical staining showed a higher expression of these proteins in ACC xenograft and clinical ACC tissue compared to NSG. Furthermore, Expression of these proteins was correlated with the histologic grading of ACC (n = 10). Therefore, our data indicate that maspin and stathmin may be not only useful biomarkers of ACC but also markers of biologic behavior in this tumor.

Animals↗

PubMed2005

Peptidomic analysis of human blood specimens: comparison between plasma specimens and serum by differential peptide display.

The human Plasma Proteome Project pilot phase aims to analyze serum and plasma specimens to elucidate specimen characteristics by various proteomic techniques to ensure sufficient sample quality for the HUPO main phase. We used our proprietary peptidomics technologies to analyze the samples distributed by HUPO. Peptidomics summarizes technologies for visualization, quantitation, and identification of the low-molecular-weight proteome (<15 kDa), the "peptidome." We analyzed all four HUPO specimens (EDTA plasma, citrate plasma, heparin plasma, and serum) from African- and Asian-American donors and compared them to in-house collected Caucasian specimens. One main finding focuses on the most suitable method of plasma specimen collection. Gentle platelet removal from plasma samples is beneficial for improved specificity. Platelet contamination or activation of platelets by low temperature prior to their removal leads to distinct and multiple peptide signals in plasma samples. Two different specimen collection protocols for platelet-poor plasma are recommended. Further emphasis is placed on the differences between plasma and serum on a peptidomic level. A large number of peptides, many of them in rather high abundance, are only present in serum and not detectable in plasma. This ex vivo generation of multiple peptides hampers discovery efforts and is caused by a variety of factors: the release of platelet-derived peptides, other peptides derived from cellular components or the clot, enzymatic activities of coagulation cascades, and other proteases. We conclude that specimen collection is a crucial step for successful peptide biomarker discovery in human blood samples. For analysis of the low-molecular-weight proteome, we recommend the use of platelet-depleted EDTA or citrate plasma.

Anticoagulants↗

PubMed2005

Pancreatic cancer proteome: the proteins that underlie invasion, metastasis, and immunologic escape.

BACKGROUND & AIMS: Pancreatic cancer is a highly lethal disease that has seen little headway in diagnosis and treatment for the past few decades. The effective treatment of pancreatic cancer is critically relying on the diagnosis of the disease at an early stage, which still remains challenging. New experimental approaches, such as quantitative proteomics, have shown great potential for the study of cancer and have opened new opportunities to investigate crucial events underlying pancreatic tumorigenesis and to exploit this knowledge for early detection and better intervention. METHODS: To systematically study protein expression in pancreatic cancer, we used isotope-coded affinity tag technology and tandem mass spectrometry to perform quantitative proteomic profiling of pancreatic cancer tissues and normal pancreas. RESULTS: A total of 656 proteins were identified and quantified in 2 pancreatic cancer samples, of which 151 were differentially expressed in cancer by at least 2-fold. This study revealed numerous proteins that are newly discovered to be associated with pancreatic cancer, providing candidates for future early diagnosis biomarkers and targets for therapy. Several differentially expressed proteins were further validated by tissue microarray immunohistochemistry. Many of the differentially expressed proteins identified are involved in protein-driven interactions between the ductal epithelium and the extracellular matrix that orchestrate tumor growth, migration, angiogenesis, invasion, metastasis, and immunologic escape. CONCLUSIONS: Our study is the first application of isotope-coded affinity tag technology for proteomic analysis of human cancer tissue and has shown the value of this technology in identifying differentially expressed proteins in cancer.

Humans↗

PubMed2026

Cryopreservation-induced proteomic alterations in Pêga donkey (Equus asinus) spermatozoa.

Semen cryopreservation is a key tool for assisted reproduction and genetic conservation, but its efficiency remains limited in donkeys, compromising post-thaw sperm quality. This study aimed to characterize the proteomic profile of Pêga donkey spermatozoa and to investigate molecular mechanisms associated with cryopreservation-induced impairment of sperm function. Semen samples were collected from Pêga jacks and evaluated for sperm motility and vigor before and after cryopreservation. Quantitative proteomic analysis was performed by LC-MS/MS, followed by bioinformatic characterization of differentially abundant proteins. Cryopreservation markedly reduced sperm motility in all animals, whereas sperm vigor showed only a non-significant tendency toward reduction, suggesting sublethal cryoinjury primarily affecting flagellar efficiency. Proteomic profiling identified 554 proteins, of which 98 were differentially abundant between in natura and cryopreserved spermatozoa. Functional enrichment analyses showed that these proteins were mainly associated with energy metabolism, mitochondrial oxidative phosphorylation, glycolysis, cytoskeletal organization, signal transduction, proteostasis, and oxidative stress response. Notably, proteins involved in ATP production, mitochondrial function, and axonemal organization were significantly altered, supporting a mechanistic link between metabolic dysfunction, flagellar structural disorganization, and reduced post-thaw motility. Overall, cryopreservation induced coordinated and compartment-specific remodeling of the donkey sperm proteome, particularly affecting pathways essential for motility and functional competence. These findings provide new molecular insights into the cryobiological vulnerability of donkey spermatozoa and establish a mechanistic basis for the development of more effective, biology-driven cryopreservation strategies for this species.

Animals↗

PubMed2003

Targeted cellular process profiling approach for uterine leiomyoma using cDNA microarray, proteomics and gene ontology analysis.

This study utilized both cDNA microarray and two-dimensional protein gel electrophoresis technology to investigate the multiple interactions of genes and proteins involved in uterine leiomyoma pathophysiology. Also, the gene ontology analysis was used to systematically characterize the global expression profiles at cellular process levels. We profiled differentially expressed transcriptome and proteome in six-paired leiomyoma and normal myometrium. Screening up to 17 000 genes identified 21 upregulated and 50 downregulated genes. The gene-expression profiles were classified into mutually dependent 420 functional sets, resulting in 611 cellular processes according to the gene ontology. Also, protein analysis using two-dimensional gel electrophoresis identified 33 proteins (17 upregulated and 16 downregulated) of more than 500 total spots, which was classified into 302 cellular processes. Of these functional profilings, downregulations of transcriptomes and proteoms were shown in cell adhesion, cell motility, organogenesis, enzyme regulator, structural molecule activity and response to external stimulus functional activities that are supposed to play important roles in pathophysiology. In contrast, the upregulation was only shown in nucleic acid-binding activity. Taken together, potentially significant pathogenetic cellular processes were identified and showed that the downregulated functional profiling has a significant impact on the discovery of pathogenic pathway in leiomyoma. Also, the gene ontology analysis can overcome the complexity of expression profiles of cDNA microarray and two-dimensional protein analysis via its cellular process-level approach. Therefore, a valuable prognostic candidate gene with relevance to disease-specific pathogenesis can be found at cellular process levels.

Cell Communication↗

PubMed2026

Takotsubo Syndrome: The First Non-Acute Proteomic Analysis by Remote Dried Blood Microsampling.

Takotsubo syndrome (TTS) is an under-recognized form of acute-onset heart failure typically precipitated by stress. While recovery of cardiac function is described over the course of weeks, adverse outcomes after apparent recovery are increasingly recognized. However, the pathophysiology of non-acute manifestations remains poorly understood. We used mass-spectrometry-based discovery proteomics from remotely collected non-acute dried blood microsamples to perform a case-control study in 62 participants with a prior TTS episode (median of 2.24 years prior to sample collection) and 47 reference controls. We quantified 398 unique proteins, and found that agnostic clustering techniques showed separation between TTS and reference control samples. This represents the first proteomic characterization of non-acute TTS. Pathway analysis of the 52 differentially regulated proteins demonstrated enrichment of proteins involved in complement activation, nitric oxide signaling, and with antioxidant activity. These enriched pathways may be suggestive of a persistent cardiomyopathy resulting from or predisposing to TTS.

Humans↗

PubMed2004

Proteomic signatures: amino acid and oligopeptide compositions differentiate among phyla.

Availability of complete genome sequences allows in-depth comparison of single-residue and oligopeptide compositions of the corresponding proteomes. We have used principal component analysis (PCA) to study the landscape of compositional motifs across more than 70 genera from all three superkingdoms. Unexpectedly, the first two principal components clearly differentiate archaea, eubacteria, and eukaryota from each other. In particular, we contrast compositional patterns typical of the three superkingdoms and characterize differences between species and phyla, as well as among patterns shared by all compositional proteomic signatures. These species-specific patterns may even extend to subsets of the entire proteome, such as proteins pertaining to individual yeast chromosomes. We identify factors that affect compositional signatures, such as living habitat, and detect strong eukaryotic preference for homopeptides and palindromic tripeptides. We further detect oligopeptides that are either universally over- or underabundant across the whole proteomic landscape, as well as oligopeptides whose over- or underabundance is phylum- or species-specific. Finally, we report that species composition signatures preserve evolutionary memory, providing a new method to compare phylogenetic relationships among species that avoids problems of sequence alignment and ortholog detection.

Amino Acid Motifs↗

PubMed2003

Identification of markers for the selection of patients undergoing renal cell carcinoma-specific immunotherapy.

Renal cell carcinoma (RCC) represents the most common malignant tumor in the kidney and is resistant to conventional therapies. The diagnosis of RCC is often delayed leading to progression and metastatic spread of the disease. Thus, validated markers for the early detection of the disease as well as selection of patients undergoing specific therapy is urgently needed. Using treatment with the monoclonal antibody (mAb) G250 as a model, proteome-based strategies were implemented for the identification of markers which may allow the discrimination between responders and nonresponders prior to application of G250-mediated immunotherapy. Flow cytometry revealed G250 surface expression in approximately 40% of RCC cell lines, but not in the normal kidney epithelium cell lines. G250 expression levels significantly varied thereby distinguishing between low, medium and high G250 expressing cell lines. Comparisons of two-dimensional gel electrophoresis expression profiles of untreated RCC cell lines versus RCC cell lines treated with a mAb directed against G250 and the characterization of differentially expressed proteins by mass spectrometry and/or Edman sequencing led to the identification of proteins such as chaperones, antigen processing components, transporters, metabolic enzymes, cytoskeletal proteins and unknown proteins. Moreover, some of these differentially expressed proteins matched with immunoreactive proteins previously identified by proteome analysis combined with immunoblotting using sera from healthy donors and RCC patients, a technique called PROTEOMEX. Immunohistochemical analysis of a panel of surgically removed RCC lesions and corresponding normal kidney epithelium confirmed the heterogeneous expression pattern found by proteome-based technologies. In conclusion, conventional proteome analysis as well as PROTEOMEX could be successfully employed for the identification of markers which may allow the selection of patients prior to specific immunotherapy.

Antibodies, Monoclonal↗

PubMed2006

In vivo proteomic analysis of the intracellular bacterial pathogen, Francisella tularensis, isolated from mouse spleen.

Understanding the pathogenesis of infectious diseases requires comprehensive knowledge of the proteins expressed by the pathogen during in vivo growth in the host. Proteomics provides the tools for such analyses but the protocols required to purify sufficient quantities of the pathogen from the host organism are currently lacking. Here, we present a rapid immunomagnetic protocol for the separation of Francisella tularensis, a highly virulent bacterium and potential biowarfare agent, from the spleens of infected mice. In less than one hour, bacteria can be isolated in quantities sufficient to carry out meaningful proteomic comparisons with in vitro grown bacteria. Furthermore, the isolates are virtually free from contaminating host proteins. Two-dimensional gel analysis revealed a host induced proteome in which 78 proteins were differentially expressed in comparison to in vitro grown controls. The results obtained clearly demonstrate the complexity of the adaptive response of F. tularensis to the host environment, and the difficulty of mimicking such behavior in vitro.

Animals↗