Browsing by Subject "proteomics"
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Item Development of a MALDI-Ion Mobility-Surface-Induced Dissociation-Time-of-flight-mass spectrometer for the analysis of peptides and proteins(Texas A&M University, 2004-09-30) Stone, Earle GregoryPeptide sequencing by surface-induced dissociation (SID) on a MALDI-Ion Mobility-orthogonal-TOF mass spectrometer is demonstrated. The early version of the instrument used for proof-of-concept experiments achieves a mobility resolution of approximately 20 and TOF mass resolution better than 200. Peptide sequences of four peptides from a tryptic digest of cytochrome c (ca. 1 pmol deposited) were obtained. The advantage of IM-SID-o-TOFMS is that a single experiment can be used to simultaneously measure the molecular weights of the tryptic peptide fragments (peptide mass mapping) and partial sequence analysis, (real time tandem mass spectrometry.) Optimization of the MALDI-IM-SID-o-TOF mass spectrometer for peptide sequencing is discussed. SID spectra obtained by using stainless steel, Au grids, and fluorinated self-assembled monolayers (F-SAM) on Au are compared. Optimum collision energies differ for the various surfaces. The fragmentation patterns observed for a series of peptides and protein digests using the Nd:YAG laser (355 nm) for MALDI ion formation and an FSAM surface for ion activation is compared to the fragmentation patterns observed for CID and photodissociation. The fragmentation patterns observed in all cases are strikingly similar. Photodissociation produced a greater abundance of ions resulting from side-chain cleavages. As a general rule optimized SID spectra contain fewer immonium ions than either photodissociation or CID. Evaluation of an instrument incorporating a new hybrid drift cell is discussed. Spectra for a digest of hemoglobin is compared to that acquired with an ABI 4700 TOF-TOF. The performance of the instrument is also evaluated using a micro-crystal Nd:YAG laser (355 nm) for MALDI operated at 400 Hz. Experiments were performed to determine the sensitivity and overall performance of the instrument. The reproducibility of the MS/MS spectra for gramicidin S is shown to be 94% run-to-run. The best mobility resolution obtained for a neat deposition of the dye Crystal Violet was 60 t/?t. Sensitivity was tested with the peptide fibrinopeptide A (m/z 1537, AA sequence ADSGEGDFLAEGGGVR). Data acquired for sixty seconds with approximately sixty femtomoles deposited. Abundant [M+H]+ ions where observed as well as [M+H]+-NH3 ions. The S/N for this short run was insufficient to identify any SID fragmentsItem Early markers of breast cancer in nipple aspirate fluid(2007-07-12) Yafei Huang; Lee-Jane W Lu; Suzanne AW Fuqua; Karl E Anderson; Jonanthan Ward; Anthony M Haag; Alexander KuroskyNipple aspirate fluid (NAF) refers to the small amount of secretion that is found in breast ducts/lobules of most non-lactating women. This fluid can be collected repeatedly and non-invasively via the nipple using a modified breast pump, and therefore, it is considered to be a potential source for identifying markers of breast cancer. The purpose of this study was to understand factors associated with the ability to secrete fluid and factors associated with the major protein profiles in NAF; and to identify protein profiles of NAF in a group of healthy non-lactating women who were 30-40 years old, not pregnant, not breastfeeding, and not taking contraceptive medications.\r\n\r\nAmong 238 women studied, 66% were secretors of NAF. Using multivariate logistic regression models, higher dietary intake of lactose [Odds Ratio (OR)=2.7; 95% Confidence Interval (CI): 1.5-4.8], earlier menarche (OR=0.8, CI: 0.7-1.0), being parous (OR=2.3, CI: 1.0-5.6), and older at first childbirth (OR=1.5, CI: 1.0-2.1) were found to be independent and positive predictors for being a secretor of NAF. These findings suggest that dietary intake of lactose, a modifiable factor, may be used to change the NAF secretor status of women. \r\n\r\nNAF were analyzed for major proteins. Two major types of protein profiles, type I and type II, were identified. Type I NAF contains proteins found in cystic disease fluid of the breast, whereas type II NAF is enriched in milk-associated proteins. Using multiple logistic regression, type I NAF was predicted independently (P<0.05) by higher body fat mass (Odds Ratio=3.0; CI: 1.5-6.1), more years since last childbirth (OR=2.6; 95% CI: 1.3-5.2) and a higher percentage of calories from saturated fat (OR=4.1; 95% CI: 1.1-14.6). These results suggest that protein profiles of NAF might be influenced by amounts and/or types of dietary and body fat. \r\n\r\nTwo different analytical strategies, 2D gel analysis coupled with MALDI-TOF/TOF, and 1D gel coupled with LC-MS/MS, were used to characterize protein profiles of type I and II NAF. Using these two strategies, a total of 99 proteins were identified: 13 unique to type I NAF, 57 unique to type II NAF, and 29 common to both types. These strategies will be used to characterize proteins in NAF of breast cancer cases. \r\nItem Proteomics of Oxidative Stress Using Inducible CYP2E1 Expressing HepG2 Cells and 3T3-L1 Adipocytes as Model Systems(2012-07-16) Newton, Billy WalkerThe overall goal of this research was to investigate oxidative stress related changes to the proteomes of 3T3-L1 adipocytes and an inducible CYP2E1 expressing HepG2 cells. Enhanced oxidative stress in hypertrophic adipocytes is associated with metabolic dysregulation and insulin resistance. Because mitochondria generate reactive oxygen species (ROS), we monitored changes to the adipocyte mitochondrial proteome during differentiation and enlargement. We labeled mitochondrial extracts from 3T3-L1 cells that were 0, 4, 7, 10, 14, and 18 days post differentiation with iTRAQ, followed by MS based identification. We found citric acid cycle proteins such as pyruvate carboxylase, citrate synthase, as well as beta-oxidation enzymes; cartinine acyl transferase and long-chain enoyl-CoA hydratase up-regulated from 7 through 18 days post differentiation onset. These data indicate TCA up-regulation for enhanced metabolic and citrate output necessary for lipid synthesis in adipocytes. Paradoxically, the data also show the simultaneous increase in the fatty acid oxidation, indicating a metabolic overdrive state. Biochemical assays showing peaks in ATP and ROS generation in 3 day old adipocytes provide further evidence of this overdrive state. A second peak in ROS generation occurred in 10 day old adipocytes; concurrent ATP generation reduced to near pre-adipocyte levels and this may indicate a metabolic shift that may be responsible for increased oxidative stress in hypertrophic adipocytes. We developed a doxycycline inducible CYP2E1 expressing HepG2 cell line using the pTet-On/pRevTRE expression system to allow greater control and sensitivity in the generation CYP2E1 mediated oxidative stress. Our cell line (RD12) demonstrated stability and tight expression control. After induction, RD12 cells showed 30 percent higher CYP2E1 activity when compared to the constitutive E47 cell line. RD12 cells showed 30 percent greater toxicity than E47 cells and 25 percent less free glutathione when exposed to 20 mM acetaminophen, indicating RD12 cells are more sensitive to the effects reactive intermediates and oxidative stress generated by CYP2E1. We conducted a survey of the toxicity of dietary fatty acids (oleic, linoleic, and palmitic) on HepG2 cells to determine fatty acid doses that induced metabolic changes, but did not cause excessive cell death. The dose of 0.20 mM linoleic and palmitic acid for 48 hours produced low toxicity, but oleic acid actually produced lower toxicity than untreated cells. After exposure cells were treated with a pro-oxidant to determine which fatty acid increased the susceptibility to protein carbonylation. The carbonylated protein isolation procedure indicated the palmitic acid may induce more carbonylation than oleic acid, but greater efficiency in the isolation procedure is required for a confidant determination.Item Wavelet methods and statistical applications: network security and bioinformatics(Texas A&M University, 2005-11-01) Kwon, DeukwooWavelet methods possess versatile properties for statistical applications. We would like to explore the advantages of using wavelets in the analyses in two different research areas. First of all, we develop an integrated tool for online detection of network anomalies. We consider statistical change point detection algorithms, for both local changes in the variance and for jumps detection, and propose modified versions of these algorithms based on moving window techniques. We investigate performances on simulated data and on network traffic data with several superimposed attacks. All detection methods are based on wavelet packets transformations. We also propose a Bayesian model for the analysis of high-throughput data where the outcome of interest has a natural ordering. The method provides a unified approach for identifying relevant markers and predicting class memberships. This is accomplished by building a stochastic search variable selection method into an ordinal model. We apply the methodology to the analysis of proteomic studies in prostate cancer. We explore wavelet-based techniques to remove noise from the protein mass spectra. The goal is to identify protein markers associated with prostate-specific antigen (PSA) level, an ordinal diagnostic measure currently used to stratify patients into different risk groups.