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PubMed2005

Proteomic method for the quantification of methionine sulfoxide.

Glycoxidation and lipoxidation reactions contribute to the chemical modification of proteins during the Maillard reaction. Reactive oxygen species, produced during the oxidation of sugars and lipids in these processes, irreversibly oxidize proteins. Methionine is particularly susceptible to oxidation, yielding the oxidation product methionine sulfoxide (MetSO). Here we describe a method for the analysis of MetSO using proteomic techniques. Using these techniques, we measured MetSO formation on the model protein RNase during aerobic incubations with glucose and arachidonate. We also evaluated the susceptibility of MetSO to reduction by NaBH4), a commonly used reductant in the analysis of Maillard reaction products.

Aerobiosis↗

PubMed2005

Effects of formaldehyde inhalation on lung of rats.

OBJECTIVE: To analyze protein changes in the lung of Wistar rats exposed to gaseous formaldehyde (FA) at 32-37 mg/m3 for 4 h/day for 15 days using proteomics technique. METHODS: Lung samples were solubilized and separated by two-dimensional electrophoresis (2-DE), and gel patterns were scanned and analyzed for detection of differently expressed protein spots. These protein spots were identified by MALDI-TOF-MS and NCBInr protein database searching. RESULTS: Four proteins were altered significantly in 32-37 mg/m3 FA group, with 3 proteins up-regulated, 1 protein down-regulated. The 4 proteins were identified as aldose reductase, LIM protein, glyceraldehyde-3-phosphate dehydrogenase, and chloride intracellular channel 3. CONCLUSION: The four proteins are related to cell proliferation induced by FA and defense reaction of anti-oxidation. Proteomics is a powerful tool in research of environmental health, and has prospects in search for protein markers for disease diagnosis and monitoring.

Administration, Inhalation↗

PubMed2006

[Proteomic analysis of human ovarian cancer cell lines and their platinum-resistant clones].

OBJECTIVE: To perform comparative proteomic analysis of human ovarian cancer cell lines for detecting platinum-resistance associated proteins. METHODS: The total proteins of two sensitive (SKOV3 and A2780) and four resistant (SKOV3/CDDP, SKOV3/CBP, A2780/CDDP and A2780/CBP) human ovarian cancer cell lines were separated by two-dimensional gel electrophoresis (2-DE). The differentially expressed proteins were analyzed using image analysis software, stained with Coomassie Brilliant Blue, then identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and database searching. The mRNA and protein levels of the differentially expressed protein which was most significant in all of the four resistant cell lines were validated by RT-PCR and western blotting, respectively. RESULTS: Five proteins were found to be significant in four cell lines. Annexin A3 and destrin were up-regulated and nicotinamide-adenine dinucleotide phosphate (NADP)-dependent isocitrate dehydrogenase 1 was down-regulated in all the four resistant samples. Glutathione transferase omega 1 had an increased expression in the other three resistant cell lines except for SKOV3/CBP in which its expression was not changed. However, cofilin 1 represented a different trend. In the two resistant sublines of SKOV3, cofilin 1 had a down-regulation, but it had an up-regulation in the cell lines induced from SKOV3. The expression of annexin A3 was up-regulated by 3 - 20 fold and the results of RT-PCR and western blotting showed complete consistency with that by 2-DE. CONCLUSIONS: Proteomic techniques are useful to the identification of the resistance-associated proteins in ovarian cancer platinum-resistant cell lines and five candidates have been found. The five differential proteins might become hopeful candidate biomarkers for resistance.

Annexin A3↗

PubMed2006

Proteomic study of plasma proteins in pregnant women with asthma.

OBJECTIVE AND BACKGROUND: The course of asthma may be altered during pregnancy with at least one-third of women experiencing a worsening of asthma and 20% having an exacerbation during pregnancy. This study used the novel proteomic technique, surface-enhanced laser desorption ionization-time of flight mass spectrometry to determine if the presence of asthma during pregnancy was associated with alterations in plasma proteins. METHODS: Plasma collected from healthy (n = 23) and asthmatic (n = 27) pregnant women at 18 and 30 weeks gestation was applied to strong anion exchange (SAX2), weak cation exchange (WCX2) and immobilized metal affinity capture (IMAC-Cu(2+)) chips. Mass analysis was conducted using Ciphergen Protein Biology System IIc and significant differences in individual peak intensities between groups determined. RESULTS: At 18 weeks gestation, 91 peaks were significantly different between pregnant women with and without asthma, representing 28% of the total peaks identified. At 30 weeks gestation, 51 peaks were significantly different. There were two peaks that were significantly different between groups at both 18 and 30 weeks gestation and expressed at a similar level at both time points. One was increased in asthmatics (MW = 6444 Da) whereas the other decreased in asthmatics compared with non-asthmatic women (MW = 1846 Da). CONCLUSIONS: This study demonstrated that there are differences in protein patterns between pregnant women with and without asthma. Other techniques are needed to define the molecular species and classify pathophysiological significance. Surface-enhanced laser desorption ionization-time of flight mass spectrometry has potential as a tool to monitor disease progression in situations such as pregnancy.

Adult↗

PubMed2001

Identification of alpha(1)-antitrypsin variants in plasma with the use of proteomic technology.

BACKGROUND: Proteomic technology permits the investigation of genetic metabolic diseases at the level of protein expression. Changes in the expression, polypeptide structure, and posttranslational modification of individual proteins can be detected in complex mixtures of proteins. METHODS: We used high-resolution two-dimensional polyacrylamide gel electrophoresis to separate isoforms of plasma proteins and detect abnormalities of mass and/or charge. We confirmed the identity of the separated proteins by in-gel digestion with proteases and N-glycanases and then analyzed the released peptides and glycans by matrix-assisted laser-desorption ionization-time-of-flight mass spectrometry. RESULTS: Complete characterization of the polypeptide sequences and glycosylation of alpha(1)-antitrypsin isoforms was achieved in plasma from controls and from patients with three different known alpha(1)-antitrypsin deficiencies and congenital disorder of glycosylation type Ia. CONCLUSIONS: This study shows that proteomic techniques are a powerful and sensitive means of detecting changes in the amino acid sequence and abnormal posttranslational modifications of specific proteins in a complex biologic matrix.

Amino Acid Sequence↗

PubMed2005

The potential of Bothrops moojeni venom in the field of hemostasis. Established use and new insights.

Early studies in the 1930s on the venom of South American Lancehead snakesofthe Bothrops genuslead to the discovery of compounds active in blood coagulation such as batroxobin and botrocetin. The scope of our investigations is to have a deeper look at the crude venom of B. moojeni using state-of-the-art proteomics methods, as well as newly developed bioassays screening for activities in the different fields of application. The proteomics techniques used up to now have included different chromatography methods, mass spectrometry, and bio-computing. The bioassays are focussed on enzymatic and other activities in the field of hemostasis and fibrinolysis. Besides the known activities several new and interesting ones have been found. They still need to be studied and confirmed in more specific supplementary assays.

Animals↗

PubMed2004

What can proteomic analyses contribute to understanding the molecular biology and clinical behavior of prostate cancer?

Identifying the proteins and their complex interactions that promote and/or sustain the aggressive malignant phenotype is essential for understanding key effectors of the molecular biology of prostate cancer. This is also essential for development of new clinical applications. A variety of proteomic techniques, ranging from mass spectrometry to new methods of multiplexing protein identification, have great potential for rapidly achieving these goals. However, in order to obtain meaningful results, these techniques must be applied within the context of our knowledge of the heterogeneity of prostate tissues and tumors, the impact of specimen processing on both the quality and quantity of proteins detected and a thorough understanding of prostate cell biology. Collaboration between the protein chemist and the prostate cell biologist will expedite progress in this important field.

Electrophoresis, Gel, Two-Dimensional↗

PubMed2006

Blood-brain barrier genomics and proteomics: elucidating phenotype, identifying disease targets and enabling brain drug delivery.

The blood-brain barrier (BBB) regulates the passage of material between the bloodstream and the brain. Recent genomic and proteomic studies of the BBB have identified some of the unique molecular characteristics of this vascular bed, and have reinforced the concept that the BBB is heavily involved in brain function. Genomic and proteomic techniques have also been used to analyze the molecular events underlying diseases that have BBB involvement, such as multiple sclerosis, Alzheimer's disease, stroke and HIV-1 infection. It is expected that a better understanding of the complex mechanisms that link the BBB to neurological disease will ultimately lead to the development of innovative treatments.

Animals↗

PubMed2003

Anti-cancer effects of butyrate: use of micro-array technology to investigate mechanisms.

Epidemiological evidence suggests that a high intake of resistant starch and NSP protects against colo-rectal cancer. The mechanisms underlying this protection are thought to be mediated by the short-chain fatty acid butyrate, which is present in the colonic lumen in millimolar concentrations as a result of bacterial fermentation of carbohydrates that have resisted digestion in the small intestine. In vitro studies have shown that butyrate displays a host of chemo-preventative properties including increased apoptosis, reduced proliferation, down regulation of angiogenesis, enhanced immunosurveillance and anti-inflammatory effects in colo-rectal cancer cell lines. However, the molecular mechanisms underlying the apparent chemo-preventative actions of butyrate are largely unknown. The evidence supporting the role of butyrate as an anti-cancer agent is reviewed, with particular emphasis on those studies that have attempted to elucidate the mechanism of action of butyrate. Our understanding of the mechanistic action of butyrate and its role in cancer prevention is likely to advance considerably in this post-genomic era with the application of genomic and proteomic technologies. Studies are described that have used gene array and proteomic techniques to investigate the response of colo-rectal cancer cells to butyrate. These pioneering studies illustrate the potential of these technologies to help characterise the molecular responses of the cancer cell to butyrate, and to define the role of butyrate (and other nutrients) in the prevention of colo-rectal cancer.

Animals↗

PubMed2006

Comparative proteomic analysis of apoptosis induced by sodium selenite in human acute promyelocytic leukemia NB4 cells.

Selenium (Se) is an essential trace element possessing anticarcinogenic properties. Sodium selenite (Na2SeO3) induced apoptosis in human acute promyelocytic leukemia (APL) cell line NB4 with dose and time dependency. In this study, proteomic techniques were used to study the apoptosis of NB4 cells induced by sodium selenite. Twenty-six downregulated and four upregulated proteins were identified, which exhibited a 1.5-fold change or greater. The identified proteins included key regulators of signal transduction such as Rho GDP dissociation inhibitor (Rho GDI) alpha and beta members of the MAPK family, and proteins involved in the regulation of c-fos or c-myc expression. Importantly, the identified proteins, hnRNP D0B and Rho GDI beta, which were related with the regulation of c-myc, c-fos, and c-jun, were determined by reverse transcription-polymerase chain reaction (RT-PCR) to confirm their downregulation in proteomic study. Western blot analysis and RT-PCR were then performed on three associated proteins: c-Myc, c-Fos, and c-Jun, and their expression were observed to be significantly downregulated. Results showed that certain regulation involved in c-myc, c-fos, and c-jun was present in the apoptosis, and the c-Myc dependent-on and Jun N-terminal kinase (JNK) pathway also play roles.

Apoptosis↗

PubMed2006

Chemical proteomics profiling of proteasome activity.

Proteolysis is a key mechanism for protein homeostasis in living cells. This process is effected by different classes of proteases. The proteasome is one of the most abundant and versatile proteases, bearing three different proteolytic active sites. The proteasome plays an important role in essential biological pathways such as antigen presentation, signal transduction, and cell-cycle control feedback loops. The aim of this work is to design novel chemical strategies for capturing, detection, identification, and quantification--in one word, profiling--the active protease fractions of interest, in cells of different phenotypes. Here, a set of chemistry-based functional proteomics techniques is demonstrated by profiling the multi-catalytic protease activities of the proteasome. Importantly, functional profiling is complementary to expression level profiling and is an indispensable parameter for better understanding of mechanisms underlying biological processes.

Blotting, Western↗

PubMed2006

Assessment of genome and proteome profiles in cocaine abuse.

Until recently, knowledge of the impact of abuse drugs on gene and protein expression in the brain was limited to less than 100 targets. With the advent of high-throughput genomic and proteomic techniques investigators are now able to evaluate changes across the entire genome and across thousands of proteins in defined brain regions and generate expression profiles of vulnerable neuroanatomical substrates in rodent and non-human primate drug abuse models and in human post-mortem brain tissue from drug abuse victims. The availability of gene and protein expression profiles will continue to expand our understanding of the short- and long-term consequences of drug addiction and other addictive disorders and may provide new approaches or new targets for pharmacotherapeutic intervention. This chapter will review gene expression data from rodent, non-human primate and human post-mortem studies of cocaine abuse and will provide a preliminary proteomic profile of human cocaine abuse and explore how these studies have advanced our understanding of addiction.

Animals↗

PubMed2004

High throughput quantitative analysis of serum proteins using glycopeptide capture and liquid chromatography mass spectrometry.

It is expected that the composition of the serum proteome can provide valuable information about the state of the human body in health and disease and that this information can be extracted via quantitative proteomic measurements. Suitable proteomic techniques need to be sensitive, reproducible, and robust to detect potential biomarkers below the level of highly expressed proteins, generate data sets that are comparable between experiments and laboratories, and have high throughput to support statistical studies. Here we report a method for high throughput quantitative analysis of serum proteins. It consists of the selective isolation of peptides that are N-linked glycosylated in the intact protein, the analysis of these now deglycosylated peptides by liquid chromatography electrospray ionization mass spectrometry, and the comparative analysis of the resulting patterns. By focusing selectively on a few formerly N-linked glycopeptides per serum protein, the complexity of the analyte sample is significantly reduced and the sensitivity and throughput of serum proteome analysis are increased compared with the analysis of total tryptic peptides from unfractionated samples. We provide data that document the performance of the method and show that sera from untreated normal mice and genetically identical mice with carcinogen-induced skin cancer can be unambiguously discriminated using unsupervised clustering of the resulting peptide patterns. We further identify, by tandem mass spectrometry, some of the peptides that were consistently elevated in cancer mice compared with their control littermates.

Animals↗

PubMed2007

Preparative denaturing isoelectric focusing for enhancing sensitivity of proteomic studies.

Substantial evidence implicates important roles for both protein phosphorylation and protein degradation in regulation of plant defense responses. Therefore, interest is growing in applying proteomics techniques to investigate these posttranscriptional changes. We have found, however, that most proteins of interest are not visible on two-dimensional (2D) gels without previous prefractionation. This chapter describes the use of preparative denaturing isoelectric focusing to enrich for proteins of specific isoelectric points before separation by 2D gels. This method significantly increases the sensitivity of 2D gel-based comparisons.

Chemical Fractionation↗

PubMed2006

Array-based proteomics: mapping of protein circuitries for diagnostics, prognostics, and therapy guidance in cancer.

The human proteome, due to the enormity of post-translational permutations that result in large numbers of isoforms, is much more complex than the genome and alterations in cancer can occur in ways that are not predictable by translational analysis alone. Proteomic analysis therefore represents a more direct way of investigating disease at the individual patient level. Furthermore, since most novel therapeutic targets are proteins, proteomic analysis potentially has a central role in patient care. At the same time, it is becoming clear that mapping entire networks rather than individual markers may be necessary for robust diagnostics as well as tailoring of therapy. Consequently, there is a need for high-throughput multiplexed proteomic techniques, with the capability of scanning multiple cases and analysing large numbers of endpoints. New types of protein arrays combined with advanced bioinformatics are currently being used to identify molecular signatures of individual tumours based on protein pathways and signalling cascades. It is envisaged that analysing the cellular 'circuitry' of ongoing molecular networks will become a powerful clinical tool in patient management.

Biomarkers, Tumor↗

PubMed2005

Functional studies of protein tyrosine phosphatases with chemical approaches.

Protein tyrosine phosphatases (PTPs) are important signaling enzymes that serve as key regulatory components in signal transduction pathways. Defective or inappropriate regulation of PTP activity leads to aberrant tyrosine phosphorylation, which contributes to the development of many human diseases. A number of PTPs have been identified as novel therapeutic targets for the treatment of various diseases. However, because PTPs can both enhance and antagonize PTK signaling, it is essential to elucidate the physiological context in which PTPs function. Assigning the functional significance of PTPs in normal physiology and in diseases remains a major challenge in cell signaling. Efficient methodologies are needed to delineate the PTP functions. One strategy is to apply chemical genetic approaches utilizing potent and selective PTP inhibitors to study the physiological roles of the PTPs in vivo. Recent work using this approach to define the roles of PTP1B in insulin- and integrin-mediated processes is discussed. Another strategy is to apply activity-based proteomic techniques to measure globally PTP activity in both normal and pathological conditions. The ability to profile the entire PTP family on the basis of changes in their activity should greatly accelerate both the assignment of PTP function and the identification of potential therapeutic targets. Recent development on the design and characterization of activity-based PTP probes is highlighted.

Animals↗

PubMed2004

[Study on serum proteome of rat endotoxemia treated by figwort root].

OBJECTIVE: To study the serum proteome of rat endotoxemia treated by figwort root (FR). METHOD: The differences of serum proteome among rats treated with lipopolysaccharide (LPS), FR, LPS + FR and saline respectively were analyzed by two-dimensional electrophoresis (2DE) assay. RESULT: The volumes of sixteen serum proteins (xPr) in LPS induced-endotoxemia group were greatly changed compared with those of the control group. Among them, the volumes of xPr 16, 19 were significantly decreased, and the volumes of xPr 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 12, 14, 18, 23 were significantly increased. When treated with FR, the volumes of xPr 1, 6, 7, 8, 9, 11, 12, 14, 18, 23 were significantly decreased, and the volumes of xPr 8, 9, 11, 12, 23, 14 were back to normal level. Two factors statistic analysis showed that FR had interaction with LPS for xPr 1, 5, 8, 10, 11, 12, 18, 19, 20, 21, 22, and FR might be the functional antagonist of LPS. We also observed that the volumes of xPr 10, 13, 15, 20, 21, 22 were found to change significantly only in FR treated group but not in LPS treated group or control group. Interestingly, the volume of xPr 13, 20, 21, 22 were increased and the volume of xPr 10, 15 were decreased. CONCLUSION: The molecular basis of therapeutic effect of FR on endotoxemia might be through the regulation of xPr 1, 6, 7, 8, 9, 11, 12, 14, 18, 23. We can use proteomic techniques to study the molecular mechanisms of diseases treated by functional Chinese herbs and the combination of different herbs is necessary for the treatment of endotoxemia, as FR can not regulated all the changed proteins induced by LPS.

Animals↗

PubMed2005

Microglial activation induced by neurodegeneration: a proteomic analysis.

Neuroinflammation mediated by microglial activation appears to play an essential role in the pathogenesis of Parkinson disease; however, the mechanisms by which microglia are activated are not fully understood. Thus, we first evaluated the effects of two parkinsonian toxicants, manganese ethylene bisdithiocarbamate (Mn-EBDC) and 1-methyl-4-phenylpyridine (MPP+), on microglial activation as well as associated dopaminergic (DAergic) neurotoxicity in primary cell culture systems. The results demonstrated that, when rat primary mesencephalic neuron-enriched or neuron-microglia mixed cultures were treated with Mn-EBDC at 2-8 microm or MPP+ at 0.25-5 microm, respectively, for 7 days, both toxicants were capable of inducing DAergic neurodegeneration as well as activating microglia via a mechanism secondary to DAergic neurodegeneration. Furthermore activated microglia subsequently enhanced DAergic neurotoxicity induced by Mn-EBDC or MPP+. Detailed scrutiny of neuron-microglia interactions identified a fraction of the conditioned media derived from a DAergic cell line treated with Mn-EBDC or MPP+ that potently activated microglia. To further define potential mediators leading to microglial activation secondary to neurodegeneration, we utilized a quantitative proteomic technique termed SILAC (for stable isotope labeling by amino acids in cell culture) to compare the protein profiles of MPP+-treated cellular fraction that mediated microglial activation as compared with controls. The search revealed numerous novel proteins that are potentially important in neurodegeneration-mediated microglial activation, a process believed to be critical in Parkinson disease progression.

1-Methyl-4-phenylpyridinium↗