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Results for “differential proteomics analysis”

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PubMed2001

Differential expression of mycobacterial proteins following phagocytosis by macrophages.

Mycobacterium tuberculosis resides within the macrophages of the host, but the molecular and cellular mechanisms of survival are poorly understood. Recent evidence suggests that the attenuated vaccine strain Mycobacterium bovis BCG is both a deletion and regulatory mutant, yet retains both its immunoprotective and intra-macrophage survival potential. In an attempt to define M. bovis BCG genes expressed during interaction with macrophages, the patterns of protein synthesis were examined by both one- and two-dimensional gel electrophoresis of BCG while inside the human leukaemic macrophage cell line THP-1. This study demonstrated that BCG expresses proteins while resident inside macrophages that are not expressed during in vitro growth in culture media or under conditions of heat shock. Western blotting analysis revealed that some of the differentially expressed proteins are specifically recognized by human M. tuberculosis-infected sera. Proteome analysis by two-dimensional electrophoresis and MS identified six abundant proteins that showed increased expression inside macrophages: 16 kDa alpha-crystallin (HspX), GroEL-1 and GroEL-2, a 31.7 kDa hypothetical protein (Rv2623), InhA and elongation factor Tu (Tuf). Identification of proteins by such a strategy will help elucidate the molecular basis of the attenuation and the vaccine potential of BCG, and may provide antigens that distinguish infection with M. tuberculosis from vaccination with BCG.

Antibodies, Bacterial↗

PubMed2006

Changes of the cortex proteome and Apolipoprotein E in transgenic mouse models of Alzheimer's Disease.

Transgenic mice carrying human Amyloid Precursor Protein mutations present amyloid plaque deposition in the brain upon aging. In this study, we characterized the changes of cortex proteome and endogenous Apolipoprotein E in these mice. Differential analysis of two-dimensional electrophoresis images revealed spots altered upon aging, transgene addition and plaque deposition. Alpha-synuclein and cytochrome oxidase polypeptide Va were up-regulated in transgenic mice. Upon aging, expression of ATP synthase alpha, alpha enolase, UMP-CMP kinase, and dihydropyrimidinase like-2 protein was modified. These proteins and their modification probably play a role in the amyloid aggregate formation in these mice.

Alzheimer Disease↗

PubMed2006

T cell epitope definition by differential mass spectrometry: identification of a novel, immunogenic HLA-B8 ligand directly from renal cancer tissue.

In this study, we describe a differential mass spectrometric technique for the immuno-proteomic analysis of the major histocompatibility complex (MHC) peptides of a renal cell carcinoma (RCC) biopsy compared with the healthy kidney tissue of the same patient after nephrectomy. Using a stable isotope labeling approach, we could directly compare and relatively quantify 43 MHC-peptide pairs, most of which were present in similar proportions on both normal kidney and tumor. Significantly, two dominant peptides of monoisotopic masses ([M+H](+)) 973.43 u and 967.59 u, respectively, were found exclusively in the tumor sample. One of these was identified as originating from heme oxygenase-1 (HO-1), a protein involved in induction of apoptosis resistance, immuno-suppression and neoangiogenesis and reported to be up-regulated in various cancer types. Moreover, the corresponding synthetic HO-1-derived peptide was shown to be immunogenic in vitro by generation of CD8+ T cell lines with peptide-specific cytolytic activity. Thus, this peptide is an example of a differentially identified T cell epitope that could be considered as a target for immunotherapy.

Aged↗

PubMedDate not supplied

Proteomic analysis of the outer membrane of Anabaena sp. strain PCC 7120.

Anabaena is a model to analyze the evolutionary development of plastids, cell differentiation, and the regulation of nitrogen fixation. Thereby, the outer membrane proteome is the place of sensing environmental differences and during plastid development, systems for intracellular communication had to be added to the proteome of this membrane. We present a protocol for the isolation of the outer membrane from Anabaena and the analysis of the proteome using different tools. 55 proteins were identified.

Anabaena↗

PubMed2006

Proteomics of cerebral injury in a neonatal model of cardiopulmonary bypass with deep hypothermic circulatory arrest.

OBJECTIVE: Concern over neurologic injury limits safe duration of deep hypothermic circulatory arrest (DHCA) in surgery for congenital cardiac disease. Proteomics is a novel and powerful technique to study global protein changes in a given protein system. Using a neonatal model of cardiopulmonary bypass with DHCA, we sought to characterize the protein changes associated with DHCA brain injury. METHODS: Ten neonatal piglets were randomized to cardiopulmonary bypass with DHCA or sham operation. DHCA animals underwent induction of bypass (100 mL x kg(-1) x min(-1)), cooling to 18 degrees C, then DHCA for 60 minutes. Animals were rewarmed to normothermia, weaned from bypass, and harvested after 30 minutes off bypass. Sham animals underwent sternotomy without further instrumentation. Plasma samples were taken before bypass and before harvest. Proteins differentially expressed in the cerebral neocortex between the 2 groups were determined by 2-dimensional differential gel electrophoresis using fluorescent cyanine dyes and mass spectrometry. A second group of 4 piglets were similarly randomized and, after the experiment, tissues underwent perfusion-fixation for histologic examination. RESULTS: Cardiopulmonary bypass with DHCA caused extensive histologic and ultrastructural cerebral injury. Proteomic analysis of cerebral cortex found 10 protein spots to be differentially expressed; 9 were identified by mass spectrometry to represent 6 proteins, including apolipoprotein A-1, neurofilament-M protein, and enolase. Decreased expression of plasma apolipoprotein A-1 was found in DHCA. CONCLUSIONS: The acute protein changes associated with cerebral injury in a neonatal model of cardiopulmonary bypass with DHCA have been characterized. These may direct further research aimed at attenuating injury seen from cardiopulmonary bypass with DHCA.

Animals↗

PubMed2006

Effects of global system for mobile communications 1800 MHz radiofrequency electromagnetic fields on gene and protein expression in MCF-7 cells.

Despite many studies over a decade, it still remains ambiguous as to the real biological effects induced by radiofrequency electromagnetic fields (RF EMF) utilized in mobile telephony. Here we investigated global gene and protein responses to RF EMF simulating the Global System for Mobile Communications (GSM) 1800 MHz signal in human breast cancer cell line MCF-7 using genomic and proteomic approaches. GeneChip analysis identified a handful of consistent changed genes after exposure to RF EMF at specific absorption rates (SAR) of up to 3.5 W/kg for 24 h. However, these differentially transcribed genes could not be further confirmed by real-time RT-PCR assay. Meanwhile, systematic proteome analysis of the MCF-7 cells revealed that a few but different proteins were differentially expressed under continuous or intermittent RF EMF exposure at SAR of 3.5 W/kg for 24 h or less, implying that the observed effects might have occurred by chance. Overall, the present study does not provide convincing evidence that RF EMF exposure under current experimental conditions can produce distinct effects on gene and protein expression in the MCF-7 cells.

Breast Neoplasms↗

PubMed2003

Proteomic analysis of androgen-regulated protein expression in a mouse fetal vas deferens cell line.

During sex differentiation, androgens are essential for development of the male genital tract. The Wolffian duct is an androgen-sensitive target tissue that develops into the epididymis, vas deferens, and seminal vesicle. The present study aimed to identify androgen-regulated proteins that are involved in development of Wolffian duct-derived structures. We have used male mouse embryos transgenic for temperature-sensitive simian virus 40 large tumor antigen at 18 d of gestation, to generate immortalized mouse fetal vas deferens (MFVD) parental and clonal cell lines. The MFVD parental and clonal cell lines express androgen receptor protein and show features of Wolffian duct mesenchymal cells. Clonal cell line MFVD A6 was selected for proteomic analysis and cultured in the absence or presence of androgens. Subsequently, two-dimensional gel electrophoresis was performed on total cell lysates. Differentially expressed proteins were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and two androgen-regulated proteins were identified as mElfin and CArG-binding factor-A (CBF-A). CBF-A and mElfin are known to bind to cytoskeletal F-actin. Both proteins appeared to be regulated by androgens at the posttranslational level, possibly involving phosphorylation. Posttranslational modification of mElfin and CBF-A by androgens may be associated with a cytoskeletal change that is involved in androgen-regulated gene expression.

Androgens↗

PubMed2026

Exogenous ABA enhances cold tolerance of Rhododendron yedoense var. poukhanense under subzero temperature: integrating physiology, transcriptome, and proteome.

Low temperature limits the growth and ornamental value of evergreen shrubs. Rhododendron yedoense var. poukhanense, an important ornamental shrub from Northeast China, frequently suffers freezing damage during winter. While exogenous abscisic acid (ABA) enhances cold tolerance in many plants, its molecular mechanisms at subzero temperatures remain poorly understood in non-model species lacking chromosome-level reference genomes. This study investigated the effects of exogenous ABA on freezing tolerance in R. yedoense var. poukhanense at -4 °C using an integrated physiological, transcriptomic, and proteomic approach. Cutting seedlings were subjected to four treatments: CK (22°C control), A (22°C + ABA), LT (-4°C), and ALT (-4°C + ABA). Photosynthetic pigments, osmotic regulation substances, antioxidant enzyme activities, and malondialdehyde (MDA) content were measured. Transcriptome sequencing and quantitative proteomics were performed, and transcriptome data were validated by quantitative real-time PCR (qRT-PCR) of 15 selected genes. ABA pretreatment reduced visible cold injury severity, partially preserved photosynthetic pigments, decreased MDA content by 28.7%, and promoted recovery of catalase (+43.6%), superoxide dismutase (+31.1%), and peroxidase (+20.0%) activities under freezing stress. Transcriptome analysis revealed 8, 444 differentially expressed genes (DEGs) in LT versus CK and 6, 481 DEGs in ALT versus CK, representing a 23% reduction in transcriptional reprogramming scope attributable to ABA priming. The ALT versus LT comparison identified only 1, 690 additional DEGs, indicating that most cold-responsive genes were pre-activated during the ABA priming phase. Proteome analysis identified 1, 461 differentially expressed proteins (DEPs) in ALT versus CK. Integrated analysis revealed extensive post-transcriptional regulation, with transcript-protein concordance of only 1.0-4.1%, and co-enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways in both omics layers. qRT-PCR validation confirmed high reliability of the transcriptome data (R2 = 0.8500). These findings demonstrate that exogenous ABA enhances freezing tolerance through multi-layered molecular regulation encompassing transcriptional buffering, translational reprogramming, and functional reallocation from photosynthesis to stress protection. This study provides the first integrated physiology-transcriptome-proteome framework for ABA-mediated freezing tolerance in an evergreen ornamental shrub and offers theoretical support for ABA-based winter protection strategies.

Rhododendron yedoense var. Poukhanense↗

PubMed2026

Exploratory proteomic and metabolomic profiling of pleural effusions identifies histone H4 and alanine as promising complementary markers for pleural tuberculosis.

The diagnosis of pleural tuberculosis (Pl-TB) remains challenging. Histopathological analysis and pathogen detection in pleural biopsies are informative but limited. We investigated differentially expressed proteins and metabolites in pleural effusions from patients with Pl-TB, malignancies, and other pathologies. A proteomic analysis of pooled pleural effusions identified 45 proteins exclusively detected or upregulated in Pl-TB samples, many linked to infectious processes. Conversely, 18 proteins were uniquely found or upregulated in malignant pleural effusions, mainly associated with detoxification and hemostasis. To validate these findings, we employed targeted proteomics in individual samples. Eight proteins were validated: S100-A9, histone H4, insulin-like growth factor-binding protein 2, fibrinogen beta chain, ficolin-3, immunoglobulin heavy constant alpha 1, sulfhydryl oxidase 1, and histidine-rich glycoprotein. Additionally, NMR-based metabolomics identified 13 metabolites with differential abundance between Pl-TB and non-TB samples. Notably, N-acetyl-glycoprotein and the branched-chain amino acids, alanine and lysine differed between groups. Proteomic and metabolomic analyses revealed distinct molecular profiles between Pl-TB and non-TB patients, despite intra-group variability. To address this, we applied classification models. Histone H4 and alanine consistently emerged as discriminative features. Overall, this study provides novel insights into the molecular landscape of Pl-TB. The combined quantification of proteins and metabolites may improve differential diagnosis, although should be further validated in larger, independent cohorts before clinical application.

Humans↗

PubMed1997

Proteome research: complementarity and limitations with respect to the RNA and DNA worlds.

A methodological overview of proteome analysis is provided along with details of efforts to achieve high-throughput screening (HTS) of protein samples derived from two-dimensional electrophoresis gels. For both previously sequenced organisms and those lacking significant DNA sequence information, mass spectrometry has a key role to play in achieving HTS. Prototype robotics designed to conduct appropriate chemistries and deliver 700-1000 protein (genes) per day to batteries of mass spectrometers or liquid chromatography (LC)-based analyses are well advanced, as are efforts to produce high density gridded arrays containing > 1000 proteins on a single matrix assisted laser desorption ionisation/time-of-flight (MALDI-TOF) sample stage. High sensitivity HTS of proteins is proposed by employing principally mass spectrometry in an hierarchical manner: (i) MALDI-TOF-mass spectrometry (MS) on at least 1000 proteins per day; (ii) electrospray ionisation (ESI)/MS/MS for analysis of peptides with respect to predicted fragmentation patterns or by sequence tagging; and (iii) ESI/MS/MS for peptide sequencing. Genomic sequences when complemented with information derived from hybridisation assays and proteome analysis may herald in a new era of holistic cellular biology. The current preoccupation with the absolute quantity of gene-product (RNA and/or protein) should move backstage with respect to more molecularly relevant parameters, such as: molecular half-life; synthesis rate; functional competence (presence or absence of mutations); reaction kinetics; the influence of individual gene-products on biochemical flux; the influence of the environment, cell-cycle, stress and disease on gene-products; and the collective roles of multigenic and epigenetic phenomena governing cellular processes. Proteome analysis is demonstrated as being capable of proceeding independently of DNA sequence information and aiding in genomic annotation. Its ability to confirm the existence of gene-products predicted from DNA sequence is a major contribution to genomic science. The workings of software engines necessary to achieve large-scale proteome analysis are outlined, along with trends towards miniaturisation, analyte concentration and protein detection independent of staining technologies. A challenge for proteome analysis into the future will be to reduce its dependence on two-dimensional (2-D) gel electrophoresis as the preferred method of separating complex mixtures of cellular proteins. Nonetheless, proteome analysis already represents a means of efficiently complementing differential display, high density expression arrays, expressed sequence tags, direct or subtractive hybridisation, chromosomal linkage studies and nucleic acid sequencing as a problem solving tool in molecular biology.

Animals↗

PubMed2006

Identification of commercial hake and grenadier species by proteomic analysis of the parvalbumin fraction.

Analysis of parvalbumin fractions through proteomic methodologies allowed the differential classification of ten commercial, closely related species of the family Merlucciidae. Muscle extracts from nine hake species of the genus Merluccius including two subspecies of Merluccius australis (australis and polylepsis) and one grenadier species Macruronus novaezelandiae with two populations (novaezelandiae and magellanicus) were evaluated by 2-DE and MALDI-TOF MS. 2-DE demonstrated that the species tested displayed a low intra-specific degree of polymorphism and the isoform patterns were noticeably species-specific. MALDI-TOF mass fingerprints showed clear differences in the pattern of peptides produced by tryptic digestion between the Merluccius and the Macruronus, making the genus differentiation possible. In addition, a selective peptide mass present in the spectra from certain hakes allowed its classification in two groups: Euro-African and American hakes. Besides, some specific masses allowed a clearly individual identification for M. bilinearis, M. australis polylepsis, M. australis australis, M. productus, M. paradoxus and M. polli, while the rest of the hake species can be grouped in two clusters, comprising M. hubbsi and M. gayi in one and M. merluccius and M. capensis in the other.

Amino Acid Sequence↗

PubMed2000

Adhesion to cellulose by Ruminococcus albus: a combination of cellulosomes and Pil-proteins?

An obligatory step in cellulose degradation by anaerobic bacteria is the adhesion of the bacterium to the polysaccharide. In many anaerobic bacteria the adhesion protein, and the enzymes required for extensive polysaccharide hydrolysis, are organized into a complex and interesting structure called the cellulosome. The Gram-positive anaerobe Ruminococcus albus also produces a cellulosome-like complex, but the bacterium appears to possess other mechanism(s) for adhesion to plant surfaces and genes encoding functions relevant to growth on cellulose are conditionally expressed, as suggested by a combination of functional proteomics, differential display reverse-transcriptase PCR, and mutational analysis. A novel form of cellulose-binding protein has been identified and shown to belong to the Pil-protein family, being most similar to the type 4 fimbrial proteins of Gram-negative, pathogenic bacteria. These studies have provided new insights into the adhesion of bacteria to plant surfaces, and call attention to the likely existence of genetically analogous adhesion determinants in both pathogenic and non-pathogenic bacteria.

Adhesins, Bacterial↗

PubMed2006

Kinase domain mutants of Bcr-Abl exhibit altered transformation potency, kinase activity, and substrate utilization, irrespective of sensitivity to imatinib.

Kinase domain (KD) mutations of Bcr-Abl interfering with imatinib binding are the major mechanism of acquired imatinib resistance in patients with Philadelphia chromosome-positive leukemia. Mutations of the ATP binding loop (p-loop) have been associated with a poor prognosis. We compared the transformation potency of five common KD mutants in various biological assays. Relative to unmutated (native) Bcr-Abl, the ATP binding loop mutants Y253F and E255K exhibited increased transformation potency, M351T and H396P were less potent, and the performance of T315I was assay dependent. The transformation potency of Y253F and M351T correlated with intrinsic Bcr-Abl kinase activity, whereas the kinase activity of E255K, H396P, and T315I did not correlate with transforming capabilities, suggesting that additional factors influence transformation potency. Analysis of the phosphotyrosine proteome by mass spectroscopy showed differential phosphorylation among the mutants, a finding consistent with altered substrate specificity and pathway activation. Mutations in the KD of Bcr-Abl influence kinase activity and signaling in a complex fashion, leading to gain- or loss-of-function variants. The drug resistance and transformation potency of mutants may determine the outcome of patients on therapy with Abl kinase inhibitors.

Amino Acid Sequence↗

PubMed2006

Early detection of lung cancer.

The overall 5-year survival of lung cancer is only 10% in Europe and 15% in the United States, and progress in curative treatments during the last 20 years has been modest. Late diagnosis of extensive disease is the main reason of failure. Early detection with low-dose spiral computed tomography (CT) is one of the most promising development of clinical research, and continuous improvements in technology can make this instrument more effective than mammography in breast cancer detection. In order to prove the benefit of early detection by reduction of lung cancer mortality, we need to enroll large numbers of high-risk individuals in multicentric prospective randomized trials combining primary prevention by smoking cessation with diagnostic intervention with low-dose spiral CT, optimal management of cancer and minimum damage for healthy individuals. Molecular biology research within early detection trials, combining genomic and proteomic analysis of blood and sputum, may improve the differential diagnosis, define the individual risk of cancer incidence and failure, and help target therapies on the basis of biologic profile.

Humans↗

PubMed2025

Proteome Profiling in Cerebrospinal Fluid Reveals Increased Levels of Peroxiredoxin 2 Discriminating Japanese Encephalitis Virus and Scrub Typhus Infection.

Japanese encephalitis virus (JEV) and scrub typhus (ST) are major etiological agents of acute encephalitis syndrome (AES) in India and South Asia. The pathophysiological changes at the molecular level caused by JEV and ST have yet to be studied in detail. The cerebrospinal fluid (CSF) proteomic landscape is a critical indicator of CNS pathology. Here, we conducted label-free quantitative proteomics on CSF from AES patients (n = 15) to identify etiology-specific differentially expressed proteins (DEPs) linked to encephalitis. The key DEPs were validated via ELISA in CSF (n = 49) and serum (n = 33). Our findings revealed 50 proteins exhibited differential expression across JEV and ST groups, with a notable subset of three proteins, PRDX2, KLK6, and TTR. PRDX2 and KLK6 were markedly elevated in JEV CSF (AUC: 0.8933 and 0.9689) but not in ST or non-JEV AES, while TTR was reduced in JEV yet elevated in ST (AUC: 0.5619 vs. 0.8238). Further, PRDX2 upregulation was validated in JEV-infected mouse brains and cortical neurons. Overexpression of PRDX2 in human neuroblastoma cells correlated with enhanced antiviral gene expression, p-STAT1, p-AKT (ser473), and viral replication. Thus, our comprehensive proteomic analysis of CSF identifies PRDX2 as an important circulatory protein, differentially expressed between JEV and ST, with high specificity and enhancing viral propagation, underscoring its role in viral propagation and pathogenesis.

Humans↗

PubMed2005

Proteomic analysis of redox- and ErbB2-dependent changes in mammary luminal epithelial cells using cysteine- and lysine-labelling two-dimensional difference gel electrophoresis.

Differential protein expression analysis based on modification of selected amino acids with labelling reagents has become the major method of choice for quantitative proteomics. One such methodology, two-dimensional difference gel electrophoresis (2-D DIGE), uses a matched set of fluorescent N-hydroxysuccinimidyl (NHS) ester cyanine dyes to label lysine residues in different samples which can be run simultaneously on the same gels. Here we report the use of iodoacetylated cyanine (ICy) dyes (for labelling of cysteine thiols, for 2-D DIGE-based redox proteomics. Characterisation of ICy dye labelling in relation to its stoichiometry, sensitivity and specificity is described, as well as comparison of ICy dye with NHS-Cy dye labelling and several protein staining methods. We have optimised conditions for labelling of nonreduced, denatured samples and report increased sensitivity for a subset of thiol-containing proteins, allowing accurate monitoring of redox-dependent thiol modifications and expression changes. Cysteine labelling was then combined with lysine labelling in a multiplex 2-D DIGE proteomic study of redox-dependent and ErbB2-dependent changes in epithelial cells exposed to oxidative stress. This study identifies differentially modified proteins involved in cellular redox regulation, protein folding, proliferative suppression, glycolysis and cytoskeletal organisation, revealing the complexity of the response to oxidative stress and the impact that overexpression of ErbB2 has on this response.

Animals↗

PubMed2005

Proteomic analysis of lysosomal acid hydrolases secreted by osteoclasts: implications for lytic enzyme transport and bone metabolism.

Osteoclasts, the bone-digesting cells, are polarized cells that secrete acid hydrolases into a resorption lacuna where bone degradation takes place. The molecular mechanisms underlying this process are poorly understood. To analyze the nature of acid hydrolases secreted by osteoclasts, we used the mouse myeloid Raw 264.7 cell line that differentiates in vitro into mature osteoclasts in the presence of the receptor activator of NF-kappaB ligand. Upon differentiation, we observed a strong increase in the secretion of mannose 6-phosphate-containing acid hydrolases. A proteomic analysis of the secreted proteins captured on a mannose 6-phosphate receptor affinity column revealed 58 different proteins belonging to several families of acid hydrolases of which 16 are clearly involved in bone homeostasis. Moreover these acid hydrolases were secreted as proproteins. The expression of most of the identified acid hydrolases is unchanged during osteoclastogenesis. Thus, our data strongly support the notion that the polarized secretion of acid hydrolases by osteoclasts results from a reorganization of key steps of membrane traffic along the lysosomal pathway rather than from a fusion of lysosomes with the membrane facing the resorption lacuna.

Animals↗

PubMed2004

Proteomic analysis for the assessment of diabetic renal damage in humans.

Renal disease in patients with Type II diabetes is the leading cause of terminal renal failure and a major healthcare problem. Hence early identification of patients prone to develop this complication is important. Diabetic renal damage should be reflected by a change in urinary polypeptide excretion at a very early stage. To analyse these changes, we used an online combination of CE/MS (capillary electrophoresis coupled with MS), allowing fast and accurate evaluation of up to 2000 polypeptides in urine. Employing this technology, we have examined urine samples from 39 healthy individuals and from 112 patients with Type II diabetes mellitus and different degrees of albumin excretion rate. We established a 'normal' polypeptide pattern in the urine of healthy subjects. In patients with Type II diabetes and normal albumin excretion rate, the polypeptide pattern in urine differed significantly from normal, indicating a specific 'diabetic' pattern of polypeptide excretion. In patients with higher grade albuminuria, we were able to detect a polypeptide pattern indicative of 'diabetic renal damage'. We also found this pattern in 35% of those patients who had low-grade albuminuria and in 4% of patients with normal albumin excretion. Moreover, we could identify several of the indicative polypeptides using MS/MS sequencing. We conclude that proteomic analysis with CE/MS permits fast and accurate identification and differentiation of polypeptide patterns in urine. Longitudinal studies should explore the potential of this powerful diagnostic tool for early detection of diabetic renal damage.

Aged↗