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PubMedDate not supplied

Application of a novel protein biochip technology for detection and identification of rheumatoid arthritis biomarkers in synovial fluid.

We compared protein profiles of the synovial fluid of patients with rheumatoid arthritis and osteoarthritis by using surface-enhanced laser desorption/ionization mass spectrometry technology. With this approach, we identified a protein expressed specifically in the synovial fluid of the patients with rheumatoid arthritis. During the investigation, we found several reproducible and discriminatory biomarker candidates for distinction between rheumatoid arthritis and osteoarthritis. Among these candidates, a 10 850 Da protein peak was the clearest example of a single signal found specifically in the rheumatoid arthritis samples. This candidate was purified using a size-exclusion spin column followed by gel electrophoresis and subsequently identified by peptide mapping and post-source decay (PSD) analysis. The results clearly indicate that the protein is myeloid-related protein 8, which was verified by the enzyme immunoassay. It is known that the myeloid-related protein 8 level in serum and synovial fluid is related to disease activity in juvenile rheumatoid arthritis. The results suggest that the ProteinChip platform is useful to detect and identify protein biomarkers expressed specifically in diseases or in some stage of diseases.

Arthritis, Rheumatoid↗

PubMed2002

Proteoglycan and proteome profiling of central human pulmonary fibrotic tissue utilizing miniaturized sample preparation: a feasibility study.

The objective of this study was to isolate fibrotic cells from human lung biopsies taken from different central pulmonary locations. A comparison was made of cell morphology, proteoglycan- and protein-expression in mesenchymal cell cultures obtained from human bronchial biopsies from patients with asthmatic-like disorders. We isolated viable cells from 10 out of the 12 biopsies. The fibroblast-like cells were positive for the biomarker a-smooth muscle actin, indicating that the cells were in an activated state. Two different types of fibroblast-like cells were observed from human pulmonary connective tissue; one of contractile type with lamellipodia that facilitate migration and a second cell type with an increased cell size, which most likely is of a synthetic phenotype. This is the first evidence of alterations in the proteoglycan expression pattern of versican, perlecan, biglycan and decorin which can be linked to the pathophysiological state of asthmatics proven by a combination of solid-phase extraction by reversed phase and by peptide mass fingerprinting using matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Protein expression analysis using two-dimensional electrophoresis was interfaced to miniaturized sample preparation techniques using microcapillary extraction. Four protein groups were identified; cytoskeletal, adhesion, scavenger and metabolic proteins. These patient's proteomes showed a high degree of heterogeneity between patients but larger homogeneity within biopsies derived from different locations of the same patient.

Actins↗

PubMed2003

Application of sensitive fluorescent dyes in linkage of laser microdissection and two-dimensional gel electrophoresis as a cancer proteomic study tool.

The combination of laser microdissection and two-dimensional gel electrophoresis (2-D PAGE) has been developed to perform proteomic analysis on specific populations of cells in cancer tissues. However, as conventional low sensitivity silver staining was used for spot detection, the microdissection required to obtain an adequate amount of protein for 2-D PAGE is laborious and only a restricted number of protein spots could be visualized. As a consequence, this technology was impractical for direct clinical applications and had a limited impact on cancer studies. To solve these problems, we developed an application in which fluorescent dyes label the proteins extracted from microdissected tissues prior to 2-D PAGE separation. In this application, a small amount of protein, less than 6.6 microg, was enough to generate a 2-D profile with approximately 1500 protein spots. This technique was applied to compare the proteome of normal intestinal epithelium with that of adenoma in Min mice. Thirty-seven protein spots reproducibly showed significant differences in intensities. Mass spectrometric analysis and Western blotting identified eight of them, including prohibitin, 14-3-3zeta, tropomyosin 3 and Hsp84. These results indicate that fluorescence labeling of proteins from microdissected tissues prior to 2-D PAGE is a powerful cancer proteomic study tool.

Adenoma↗

PubMed2003

Proteome analysis on an early transformed human bronchial epithelial cell line, BEP2D, after alpha-particle irradiation.

To probe the mechanism of carcinogenesis of lung cancer at the molecular level and to find potential protein markers involved in the early phase of tumorgenesis, differential proteome analysis on primary passage cell line R15H, and early transformed cell line R15H20 derived from (238)Pu alpha-particle irradiation of human papillomavirus (HPV) 18-immortalized human bronchial epithelial cell line (BEP2D), was carried out using two-dimensional electrophoresis (2-DE) and peptide mass fingerprinting (PMF) with matrix-assisted laser desorption/ionisation-time of flight mass spectrometry. Image analysis and Student's t-test (p < 0.05) showed that three protein spots were only expressed in R15H, intensities of 43 protein spots on the gels were altered between R15H and R15H20. Two of the three spots that were only expressed in R15H were identified as high mobility group protein 1. Two proteins decreased in abundance in R15H20 were identified as maspin precursor, a tumor suppressor and aminoacylase-1. Ornithine aminotransferase and peptidyl-prolyl cis-trans isomerase A that were increased in R15H20, were also identified. Relationships between these differentially expressed proteins and the carcinogenesis mechanism of lung cancer are discussed. The protein expression profile of the R15H cell line was also constructed during the study as a reference map for further comparative proteome analysis of the irradiation induced BEP2D cell line. Of the 90 spots analyzed with PMF in the 2-DE gel of R15H cell line, 50 proteins were identified by searching the nonredundant protein database SWISS-PROT/TrEMBL.

Alpha Particles↗

PubMed2002

Ewing sarcoma family of tumors express adenovirus receptors and are susceptible to adenovirus-mediated oncolysis.

PURPOSE: Attenuated viruses derived from adenoviruses (Ad) that kill tumor cells (oncolysis) are currently in clinical trials for selected cancers. Some cancers have proven resistant to Ad infection due to low expression of viral receptors. The authors sought to determine whether members of the Ewing sarcoma family of tumors (ESFTs) express Ad receptors and are sensitive to Ad-mediated oncolysis. METHODS: Using flow cytometry, the authors tested a panel of cell lines derived from ESFTs for expression of both the Ad receptor, coxsackie-adenovirus receptor (CAR), and the cellular mediator of Ad uptake, alpha(v)-integrins, as well as for Ad-mediated gene transduction. Cell survival assays were used to assess the sensitivity to Ad-mediated oncolysis. Immunohistochemistry was used to assess CAR expression in primary tumors. mRNA levels of CAR in cell lines and tumor samples were also queried from a cDNA expression database. RESULTS: The ESFT cell lines expressed CAR and alpha(v)-integrins, showed high levels of gene transduction, and were highly sensitive to viral oncolysis. Primary tumor samples were positive for CAR expression by immunohistochemistry. Microarray analysis confirmed CAR expression in ESFT cell lines and tumors. CONCLUSIONS: Ewing sarcoma cells express the Ad receptors and are sensitive to Ad oncolysis. Treatment of Ewing sarcoma using conditionally replicative adenoviruses should be explored.

Adenoviridae↗

PubMed2003

Proteomics: advanced technology for the analysis of cellular function.

Proteomics developed initially from the decade-long study of comprehensive protein visualization on two-dimensional electrophoresis gels has been expanded by mass spectrometry and the growth in searchable sequence databases. Currently, by use of more sophisticated technology such as a combination of multidimensional chromatography and mass spectrometry, thousands of proteins can automatically be identified in a day along with semiquantitative information on differential-protein expression. As with differential gene expression by cDNA-chips, the differential-protein analysis is useful for monitoring and identifying proteins involved in various physiological changes in cells or organisms, although the analysis alone does not necessarily provide information regarding the cause of the change or the function of the proteins. However, proteomics also provides the tools to expand into more sophisticated biochemical approaches, such as the study of protein interactions that can be determined directly by performing a pull-down assay with a bait protein followed by mass spectrometric identification of the bound proteins. Proteomics, thus, is useful for both large-scale surveys of proteins and detailed studies of the functional relationships among the proteins of interest. Certainly this approach can be applicable to the assessment of amino acid adequacy and safety.

Animals↗

PubMed2002

High-throughput cell analysis using multiplexed array technologies.

The desire for more biologically relevant data from primary screening has resulted in a dramatic increase of cell-based assays in HTS labs. Consequently, new cell-array technologies are being developed to increase the quality and quantity of data emerging from such screens. These technologies take the form of both positional and non-positional formats, each with their own advantages. Notably, screens using these technologies generate databases of high-quality data that can be analyzed in ways currently not possible. The power of cell-based assays combined with new array and analytical technologies will enable the condensation of serial drug discovery processes, thereby decreasing the time and cost of taking a hit compound into clinical trials. Here, we compare array strategies being developed towards the goal of integrating multiplexed cell-based assays into HTS.

Animals↗

PubMed2002

On-chip microextraction for proteomic sample preparation of in-gel digests.

Despite the high sensitivity and relatively high tolerance for contaminants of matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) there is often a need to purify and concentrate the sample solution, especially after in-gel digestion of proteins separated by two-dimensional gel electrophoresis (2-DE). A silicon microextraction chip (SMEC) for sample clean-up and trace enrichment of peptides was manufactured and investigated. The microchip structure was used to trap reversed-phase chromatography media (POROS R2 beads) that facilitates sample purification/enrichment of contaminated and dilute samples prior to the MALDI-TOF MS analysis. The validity of the SMEC sample preparation technique was successfully investigated by performing analysis on a 10 nM peptide mixture containing 2 m urea in 0.1 m phosphate-buffered saline with MALDI-TOF MS. It is demonstrated that the microchip sample clean-up and enrichment of peptides can facilitate identification of proteins from 2-DE separations. The microchip structure was also used to trap beads immobilized with trypsin, thereby effectively becoming a microreactor for enzymatic digestion of proteins. This microreactor was used to generate a peptide map from a 100 nM bovine serum albumin sample.

Electrophoresis, Gel, Two-Dimensional↗

PubMedDate not supplied

ProteinChip technology: a new and facile method for the identification and measurement of high-density lipoproteins apoA-I and apoA-II and their glycosylated products in patients with diabetes and cardiovascular disease.

This paper describes a ProteinChip technology for the identification and quantification of apolipoprotein profiles in crude biological samples. Expression levels of apoA-I and apoA-II and their glycosylated products were accomplished using single 1 microL plasma samples. In the present studies, strong anionic and weak cationic exchanger ProteinChips (SAX2 and WCX2 chip surfaces) were tested, and the WCX2 chip was found to be selective for specific apolipoproteins. Using the WCX2 chip and analysis via surface-enhanced laser desorption ionization mass spectrometry (SELDI-MS), apoA-I and apoA-II were separated as sharp peaks at 28 and 17 kD and did not overlap with other serum protein peaks. Since these assays can be completed on a large number of clinical samples in approximately 1 h, further development of this technique will facilitate both epidemiological studies and therapeutic trials in assessing the role of the apolipoproteins and their glycosylated products in atherosclerosis.

Apolipoprotein A-I↗

PubMed2002

Amelioration of ozone-induced lung injury by anti-tumor necrosis factor-alpha.

Ozone (O(3)) is a significant component of atmospheric air pollution and produces detrimental effects in the lung. Although the mechanism of O(3)-induced lung inflammation and injury is unclear, the increased release of the proinflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) by lung cells following O(3) exposure may shed some light on this subject. To investigate the role of TNF-alpha in the O(3)-induced pulmonary insult, we intraperitoneally injected rats with either rabbit preimmune serum or rabbit antirat TNF-alpha 1 h prior to O(3) exposure. Approximately 12 h after the end of O(3) exposure the animals were sacrificed, the lungs lavaged, and tissue samples collected for expression of cytokine genes relevant to inflammation. The bronchoalveolar lavage fluid (BALF) was analyzed for albumin as a marker of pulmonary epithelial permeability changes and for fibronectin for its role in lung injury and repair. The lavage cells were collected, counted, and identified to quantitate the inflammatory response. Ozone exposure resulted in a significant increase in BALF albumin and fibronectin as compared to air-exposed controls and a significant increase in BALF polymorphonuclear leukocytes (PMNs). Antibody treatment produced a significant decrease in BALF albumin and PMNs as compared to O(3)-exposed rats given preimmune serum. Antibody treatment did not affect the BALF fibronectin concentration or the total cell count in the BAL. Tissue analysis for gene arrays revealed an activation of IL-1alpha, IL-6, and IL-10 in animals exposed to O(3). The gene expression was downregulated in animals treated with anti-TNF-alpha antibody prior to O(3) exposure. The results suggest a central role for TNF-alpha in the mechanistic pathways critical to lung inflammation. The significance of TNF-alpha in the inflammation and epithelial injury produced by ozone exposure reflects its overall contribution through modulation of other cytokines.

Albumins↗

PubMed2003

Novel human and mouse genes encoding a shank-interacting protein and its upregulation in gastric fundus of W/WV mouse.

BACKGROUND AND AIMS: A division of labor exists between different classes of interstitial cells of Cajal (ICC) in the gastrointestinal tract. In the stomach and small intestine, ICC at the level of the myenteric plexus (IC-MY) act as slow wave pacemaker cells, whereas intramuscular ICC (IC-IM) in the stomach act as intermediaries in enteric motor neurotransmission. The muscle layers of the gastric fundus do not have IC-MY, therefore electric slow waves are not generated. Intramuscular ICC are absent in the gastric fundus of W/WV mutant mice, and excitatory and inhibitory motor nerve responses are reduced in these tissues. The absence of IC-IM in W/WV mutants in the fundus provides a unique opportunity to study the molecular changes that are associated with the loss of these cells. METHODS: The tissue gene expression of wild-type and W/WV mice from gastric fundus was assayed using a murine microarray chip analysis displaying a total of 8734 elements. RESULTS: Twenty-one queries were differentially expressed in wild-type and W/WV mice. One candidate gene, encoding a novel protein homologous to rat Shank-interacting protein (Sharpin) was significantly upregulated in fed and starved W/WV mice. The full-length clone of the murine gene and its human counterpart were isolated and designated as Shank-interacting protein-like 1 (SIPL1). Human SIPL1 complementary DNA encodes a protein of 345 amino acids. This gene was localized to chromosome 8. SIPL1 was abundantly expressed in human stomach and small intestine, and scarcely expressed in cecum and rectum. CONCLUSIONS: Gene analysis showed that SIPL1 differentially express in the gastric fundus of normal and W/WV mice. The upregulation of SIPL1 in the fundus of W/WV mice, and expression in the upper gastrointestinal tract suggest that the SIPL1 gene could be associated with ICC function in mice and humans.

Amino Acid Sequence↗

PubMed2003

Proteomic analyses of arsenic-induced cell transformation with SELDI-TOF ProteinChip technology.

In this study, we demonstrated that low levels (1.5 microM) of arsenite induces B[a]P-treated lung cell transformation. We then used a proteomic approach to identify protein expression by ProteinChips, which could potentially be important for transformation induced by this toxic metal. Most of the protein peaks in cell extracts of all samples, including the control, B[a]P-treated, and B[a]P + As-treated cells are identical. However, surface-enhanced laser desorption/ionization time of flight (SELDI-TOF) analysis with Cu-ProteinChips and WCX-ProteinChips revealed several dramatically different protein peaks that appeared in lung cells after being transformed by a treatment of 1.5 microM arsenite for 12 weeks. SAX2 ProteinChip also identified a prominent protein peak that was preferentially expressed in control cells. Interestingly, by using a SAX2 chip, we were able to detect several protein peaks that increased their expression in lung epithelial cells (LEC) treated with only B[a]P. Identification and characterization of these proteins may reveal the molecular basis of As-induced cell transformation and provide insight into the mechanisms by which arsenic induces carcinogenesis.

Animals↗

PubMed2002

Parallel analysis with optically gated sample introduction on a multichannel microchip.

As an alternative to the T-type injection on microchips, optically gated sample introduction previously has been demonstrated to provide fast, serial, and reproducible injections on a single-channel microchip. Here, the ability to perform high throughput, multichannel analysis with optically gated sample introduction is described using a voice coil actuator. The microchip is fixed on a stage, which moves back and forth via the voice coil actuator, scanning two laser beams across the channels on the microchip. For parallel analysis on a multichannel microchip, both the gating beam and the probe beam are scanned at 10 Hz to perform multiple injections and parallel detection. Simultaneous, fast separations of 4-choloro-7-nitrobenzofurazan (NBD)-labeled amino acids are demonstrated in multiple channels on a microchip. Serial separations of different samples in multiple channels are also reported. Optically gated sample introduction on multiple, parallel channels shows the potential for high-speed, high-throughput separations that are easily automated by using a single electronic shutter.

4-Chloro-7-nitrobenzofurazan↗

PubMed2003

A novel experimental design for comparative two-dimensional gel analysis: two-dimensional difference gel electrophoresis incorporating a pooled internal standard.

The comparison of two-dimensional (2-D) gel images from different samples is an established method used to study differences in protein expression. Conventional methods rely on comparing images from at least 2 different gels. Due to the high variation between gels, detection and quantification of protein differences can be problematic. Two-dimensional difference gel electrophoresis (Ettan trade mark DIGE) is an emerging technique for comparative proteomics, which improves the reproducibility and reliability of differential protein expression analysis between samples. In the application of DIGE different samples are labelled with mass and charge matched spectrally resolvable fluorescent dyes and are then separated on the same 2-D gel. Using an Escherichia coli lysate "spiked" with varying amounts of four different known proteins, we have tested a novel experimental design that exploits the sample multiplexing capabilities of DIGE, by including a standard sample in each gel. The standard sample comprises equal amounts of each sample to be compared and was found to improve the accuracy of protein quantification between samples from different gels allowing accurate detection of small differences in protein levels between samples.

Electrophoresis, Gel, Two-Dimensional↗

PubMed2003

Estradiol control of expression and levels of estradiol-binding proteins in the medial preoptic area, medial hypothalamus and pituitary.

The brains of mammals have at least three estradiol-binding proteins: estradiol receptor-alpha (ERalpha), ERbeta, and sex hormone-binding globulin (SHBG). In this study we compare the effects of estradiol treatment on the expression of mRNA for these three estradiol-binding proteins in two reproductively important brain areas, the medial preoptic area-anterior hypothalamus (MPOA-AH) and medial hypothalamus (MH) as well as in the hippocampus in ovariectomized rats, using the reverse transcriptase-polymerase chain reaction (RT-PCR). We also used surface-enhanced laser desorption ionization time of flight (SELDI-TOF) mass spectrometry (MS) to analyze the effects of estradiol in ovariectomized rats on SHBG levels in the MPOA-MH as well as the neurohypophysis. In vivo estradiol treatment in ovariectomized rats eliminated or significantly reduced expression of all three estradiol-binding proteins in both the MPOA-AH and MH. This change in ERalpha, ERbeta, and SHBG expression did not occur in the hippocampus. Both Northern blot and DNA sequence analysis confirmed the results of the RT-PCR for SHBG. SELDI-TOF MS analysis demonstrated that in vivo estradiol treatments resulted in dramatically decreased levels of SHBG in the hypothalamus and that a reduction in SHBG mRNA by estradiol treatment also resulted in a reduction in SHBG protein levels. Estradiol treatment also eliminated detectable SHBG from the neurohypophysis, suggesting that estradiol controls SHBG levels in this release site. That in vivo estradiol treatments had the same inhibitory effects on mRNA levels for SHBG and both ERs suggests similar translational control mechanisms for all three steroid-binding proteins in the brain. That estradiol treatments also reduced pituitary SHBG suggests that such treatment releases SHBG from the neurohypophysis.

Animals↗

PubMed2003

Proteomic analyses in Waldenstrom's macroglobulinemia and other plasma cell dyscrasias.

The proteomic analysis of tumor cells emerges as a key complement to gene expression profiling, primarily because regulation of protein expression (at the translational and post-translational levels) can buffer the magnitude of changes occurring at the gene transcription level, in order to fine tune cellular functions. Herein we describe the concept of proteomic analysis of the signaling state of tumor cells, as well as its application in the study of signaling pathways in plasma cell dyscrasias, such as Waldenstrom's macroglobulinemia (WM) and multiple myeloma (MM). Comparative studies of WM versus MM cells at baseline and in the setting of drug treatment reveals proteomic profiles of the signaling state with significant overlap (that could reflect a putative B-cell lineage-related proteomic signature), but also distinct differences, possibly associated with differential features in the biologic behavior and drug sensitivity of these diseases. These proteomic studies pave the way for a more comprehensive insight into the molecular basis of WM versus other B-cell malignancies.

Electrophoresis, Gel, Two-Dimensional↗

PubMed2003

Growth factor array fabrication using a color ink jet printer.

We have developed a novel method for growth factor analysis using a commercial color ink jet printer to fabricate substrata patterned with growth factors. We prepared substrata with insulin printed in a simple pattern or containing multiple areas of varying quantities of printed insulin. When we cultured the mouse myoblast cell line, C2C12, on the insulin-patterned substrata, the cells were grown in the same pattern with the insulin-printed pattern. Cell culture with the latter substrata demonstrated that quantity control of insulin deposition by a color ink jet printer is possible. For further applications, we developed substrata with insulin-like growth factor-I (IGF-I) and basic fibroblast growth factor (bFGF) spotted in 16 different areas in varying combinations and concentrations (growth factor array). With this growth factor array, C2C12 cells were cultured, and the onset of muscle cell differentiation was monitored for the expression of the myogenic regulator myogenin. The ratio of cells expressing myogenin varied with the doses of IGF-I and bFGF in the sections, demonstrating a feasibility of growth factor array fabrication by a color ink jet printer. Since a printer manipulates several colors, this method can be easily applied to multivariate analyses of growth factors and attachment factors affecting cell growth and differentiation. This method may provide a powerful tool for cell biology and tissue engineering, especially for stem cell research in investigating unknown conditions for differentiation.

Animals↗

PubMed2003

A data review and re-assessment of ovarian cancer serum proteomic profiling.

BACKGROUND: The early detection of ovarian cancer has the potential to dramatically reduce mortality. Recently, the use of mass spectrometry to develop profiles of patient serum proteins, combined with advanced data mining algorithms has been reported as a promising method to achieve this goal. In this report, we analyze the Ovarian Dataset 8-7-02 downloaded from the Clinical Proteomics Program Databank website, using nonparametric statistics and stepwise discriminant analysis to develop rules to diagnose patients, as well as to understand general patterns in the data that may guide future research. RESULTS: The mass spectrometry serum profiles derived from cancer and controls exhibited numerous statistical differences. For example, use of the Wilcoxon test in comparing the intensity at each of the 15,154 mass to charge (M/Z) values between the cancer and controls, resulted in the detection of 3,591 M/Z values whose intensities differed by a p-value of 10-6 or less. The region containing the M/Z values of greatest statistical difference between cancer and controls occurred at M/Z values less than 500. For example the M/Z values of 2.7921478 and 245.53704 could be used to significantly separate the cancer from control groups. Three other sets of M/Z values were developed using a training set that could distinguish between cancer and control subjects in a test set with 100% sensitivity and specificity. CONCLUSION: The ability to discriminate between cancer and control subjects based on the M/Z values of 2.7921478 and 245.53704 reveals the existence of a significant non-biologic experimental bias between these two groups. This bias may invalidate attempts to use this dataset to find patterns of reproducible diagnostic value. To minimize false discovery, results using mass spectrometry and data mining algorithms should be carefully reviewed and benchmarked with routine statistical methods.

Artificial Intelligence↗