Proteomics Center publications (MU)
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Items in this collection are public presentations made by Proteomics Center faculty, staff, and students, either alone or as co-authors, and which may or may not have been published in an alternate format. Items may contain more than one file type.
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Item Kinetics and mechanism of protein tyrosine phosphatase 1B inactivation by acrolein(American Chemical Society, 2007) Seiner, Derrick R., 1981-; LaButti, Jason N., 1972-; Gates, Kent S. (Kent Stephen), 1962-Human cells are exposed to the electrophilic [alpha],[beta]-unsaturated aldehyde acrolein from a variety of sources. The reaction of acrolein with functionally critical protein thiol residues can yield important biological consequences. Protein tyrosine phosphatases (PTPs) are an important class of cysteine-dependent enzymes whose reactivity with acrolein previously has not been well-characterized. These enzymes catalyze the dephosphorylation of phosphotyrosine residues on proteins via a phosphocysteine intermediate. PTPs work in tandem with protein tyrosine kinases to regulate a number of critically important mammalian signal transduction pathways. We find that acrolein is a potent time-dependent inactivator of the enzyme PTP1B (kinact = 0.02 [plus or minus] 0.005 s-1 and KI = 2.3 [plus or minus] 0.6 x 10-4 M). The enzyme activity does not return upon gel filtration of the inactivated enzyme, and addition of the competitive phosphatase inhibitor vanadate slows inactivation of PTP1B by acrolein. Together, these observations suggest that acrolein covalently modifies the active site of PTP1B. Mass spectrometric analysis reveals that acrolein modifies the catalytic cysteine residue at the active site of the enzyme. Aliphatic aldehydes such as glyoxal, acetaldehyde, and propanal are relatively weak inactivators of PTP1B under the conditions employed here. Similarly, unsaturated aldehydes such as crotonaldehyde and 3-methyl-2-butenal bearing substitution at the alkene terminus are poor inactivators of the enzyme. Overall, the data suggest that enzyme inactivation occurs via conjugate addition of the catalytic cysteine residue to the carbon-carbon double bond of acrolein. The results indicate that inactivation of PTPs should be considered as a possible contributor to the diverse biological activities of acrolein and structurally related α,β-unsaturated aldehydes.Item Development and assessment of scoring functions for protein identification using PMF data(Wiley-VCH, 2007) Song, Zhao, 1978-; Chen, Luonan; Ganapathy, Ashwin; Wan, Xiu-Feng; Brechenmacher, Laurent; Tao, Nengbing; Emerich, David W. (David William); Stacey, Gary, 1951-; Xu, Dong, 1965-PMF is one of the major methods for protein identification using the MS technology. It is faster and cheaper than MS/MS. Although PMF does not differentiate trypsin-digested peptides of identical mass, which makes it less informative than MS/MS, current computational methods for PMF have the potential to improve its detection accuracy by better use of the information content in PMF spectra. We developed a number of new probability-based scoring functions for PMF protein identification based on the MOWSE algorithm. We considered a detailed distribution of matching masses in a protein database and peak intensity, as well as the likelihood of peptide matches to be close to each other in a protein sequence. Our computational methods are assessed and compared with other methods using PMF data of 52 gel spots of known protein standards. The comparison shows that our new scoring schemes have higher or comparable accuracies for protein identification in comparison to the existing methods. Our software is freely available upon request. The scoring functions can be easily incorporated into other proteomics software packages.Item Proteomic identification of PKC-mediated expression of 20E-induced protein in Drosophila melanogaster(ACS, 2007) Sun, Yaning, 1978-; An, Shiheng; Henrich, Vincent C.; Sun, Xiaoping; Song, QishengEcdysone receptor (EcR) and its heterodimeric partner, ultraspiracle protein (USP), are nuclear receptors that mediate the action of the insect molting hormone 20-hydroxyecdysone (20E). There is evidence that the activity of both receptors is affected by phosphorylation. Using a proteomic approach, we have shown that protein kinase C (PKC) activity is necessary for mediating 20E-induced expression of 14 specific proteins, including three previously reported 20E responsive proteins, and is also responsible for the intracellular localization of EcR and USP in larval salivary glands of Drosophila melanogaster. The 20E-dependent expression of the proteins was verified using real-time PCR and/or Western blot analysis. For some genes, inhibition of PKC activity reduced 20E-dependent transcriptional activity rapidly, raising the possibility that these are direct gene targets of EcR and USP. The data further indicate that PKC-mediated phosphorylation is also required for genes regulated indirectly by 20E-induced changes in the larval salivary gland.Item Proteomics of canine lymphoma identifies potential cancer-specific protein markers(American Association for Cancer Research, 2007) McCaw, Dudley L.; Chan, Arvan S.; Stegner, Andrew L.; Mooney, Brian P.; Bryan, Jeffrey N.; Turnquist, Susan E.; Henry, Carolyn J.; Alexander, Hannah, 1947-; Alexander, Stephen, 1948-Purpose: Early diagnosis of cancer is crucial for the success of treatment of the disease, and there is a need for markers whose differential expression between disease and normal tissue could be used as a diagnostic tool. Spontaneously occurring malignancies in pets provide a logical tool for translational research for human oncology. Lymphoma, one of the most common neoplasms in dogs, is similar to human non-Hodgkin's lymphoma and could serve as an experimental model system. Experimental Design: Thirteen lymph nodes from normal dogs and 11 lymph nodes from dogs with B-cell lymphoma were subjected to proteomic analysis using two-dimensional PAGE separation and matrix-assisted laser desorption/ionization time-of-flight analysis. Results: A total of 93 differentially expressed spots was subjected to matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry analysis, and several proteins that showed differential expression were identified. Of these, prolidase (proline dipeptidase), triosephosphate isomerase, and glutathione S-transferase were down-regulated in lymphoma samples, whereas macrophage capping protein was up-regulated in the lymphoma samples. Conclusions: These proteins represent potential markers for the diagnosis of lymphoma and should be further investigated in human samples for validation of their utility as diagnostic markers.Item Guest and ligand behavior in zinc-seamed pyrogallol[4]arene molecular capsules(Wiley-VCH, 2007) Power, Nicholas P.; Dalgarno, Scott J.; Atwood, J. L.Not just an inside job: Encapsulated and confined in an octanuclear zinc-seamed pyrogallol[4]arene molecular capsule, a guest of choice can act as a reporter for electronic communication between the exterior and the interior of the capsule through ligand exchange (see structure of capsule, Zn light blue, O red, S yellow).