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Research Interests
Our main research interests are in the area of mass spectrometry and proteomics. The focus of our lab is in two main directions: 1) proteomics-based analysis of proteins, post-translational modifications of proteins and protein-protein interactions and 2) investigation of Tumor Differentiation Factor (TDF) and Jumping Translocation Breakpoint (JTB) proteins. Many proteomics projects are either underway or just started. Other emerging projects are in the area of biomarker discover, particularly Autism, Autism Spectrum Disorders, Neurodevelopmental disorders (i.e. depression), as well as breast cancer, biochemical events upon environmental exposure, etc. Some of these projects include:


Functional Investigation of Tumor Differentiation Factor. Tumor differentiation factor (TDF) is a protein produced by the pituitary gland and secreted into the blood stream, with a potential endocrine role. We hypothesize that 1) TDF protein has cellular and secreted isoforms with different functions and 2) Cellular TDF is a developmental protein important for the formation and function of the central nervous system. Therefore we plan to structurally and functionally characterize TDF. To do so, we will isolate the two TDF isoforms and characterize them for amino acid sequence, post-translational modifications and protein-protein interactions. We will also investigate the TDF-loss of function (TDF knock-down) in NG108 neuroblastoma cells and TDF gain of function (TDF over-expression) in NG108 cells and in GS9L astrocytoma cells. The differences between the proteomes of control cells and loss of function cells, as well as the proteomes of control cells and gain of function cells will be investigated by nanoLC-MS/MS. Finally, we will investigate TDF homologue from zebrafish by immunoaffinity purification and nanoLC-MS/MS.


Functional Investigation of Jumping Translocation Breakpoint (JTB). Human JTB (hJTB) is a gene located on the human chromosome 1 at q21 which is involved in the unbalanced translocation in various types of cancer. It encodes a conserved transmembrane protein of 16.4 kDa. JTB protein is ubiquitously present in normal cells and is overexpressed in various types of cancer including prostate and breast cancer. Hence this protein could be a biomarker for tumor malignancies and a potential target for their treatment. However, the pathway through which this protein causes increased cell growth and differentiation is not clear. Investigation and comparison of the proteomes of cancer cells with upregulated and downregulated JTB can be a good approach to understand the function of the protein and its contribution to tumorigenesis. Here, we plan to transfect MCF 7 breast cancer cell lines with the sense orientation of JTB cDNA in HA, His and FLAG tagged CMV expression vector to overexpress hJTB as well as use a CRISPR plasmid to knockout the hJTB gene. The transfected cells will be selected and the proteins will be extracted. We will perform western blotting by using Anti-JTB antibody to confirm the expression of JTB. The extracted proteins will also be separated by SDS-PAGE and the individual gel bands will be trypsin digested and the peptides will be analyzed by a NanoAcquity UPLC coupled with QTOF Xevo Mass Spectrometer. These studies could help us elucidate the mechanism through which JTB induces cell proliferation and test the JTB protein as a potential drug target for malignancies with overexpression of the protein.


Proteomic analysis of sera and saliva from children with Autism Spectrum Disorder (ASD) versus matched controls and correspondence with behavioral measures. Autism spectrum disorders (ASD) are highly prevalent and increasing in incidence (1 in 64 children). ASD diagnosis often occurs only after symptoms are readily apparent. Extensive research has demonstrated that better behavioral outcomes occur when ASD treatment is initiated as early as possible. An easy method of detecting ASD in young, pre-verbal children would allow earlier and more effective treatment. Such detection could be accomplished using mass spectrometry and proteomic analysis of bodily fluids such as sera or saliva. Proteomics profiling of sera and saliva may help us understand the etiology of ASD. Therefore, we believe that detection of ASD in children through proteomics-based investigation of sera and saliva for identification of biomarkers would be a prime option. Our objective is to analyze the sera and saliva from children with ASD and age-matched normal controls using mass spectrometry and correlate any identified protein changes with social impairment measures (Social Skills Improvement System, SSIS). We also hypothesize that in the sera and saliva of children with ASD, cholesterol and associated carrier molecules are abnormal and that the magnitude of abnormality will correspond with ASD severity on social measures. This project is led by Dr. Alisa G. Woods, who bridges Biochemistry & Proteomics (our lab), Psychology (Dr. Jeanne Ryan, SUNY Plattsburgh), Biochemistry (Dr. Dudley, University of Swansea, United Kingdom), Psychiatry (Dr. Thome, University of Rostock, Germany) and Child Psychiatry (Drs. Taurines & Gerlach, University of Wuerzburg, Germany).


Proteomics of human breast milk in women with breast cancer and matched controls. Breast cancer arises from cells with genetic and "epigenetic" changes. The epigenetic, or nonsequence changes, are important because they disrupt the function of genes, allowing cells to grow out of control. One type of epigenetic change called "hypermethylation" is considered one of the most promising biomarkers for assessing breast cancer risk. Most importantly, methylation changes are potentially reversible and drugs targeting methylation are in clinical trials. This project is led by Drs. Kathleen Arcaro and Brian Pentecost, from the University of Massachusetts at Amherst, who study breast cancer risk and promoter hypermethylation in breast cells obtained from the milk of nursing women. The team from Clarkson University is be responsible for biochemical fractionation and mass spectrometric identification of proteins from the human breast milk, data analysis and interpretation, as well as data validation. The end goal is to identify differences between the proteins, protein-protein interactions and post translation modifications within the breast milk samples from healthy women versus women with breast cancer.


Wisconsin’s Assessment of Healthy Consumption of Great Lakes Fish (PI Dr. Bernard Crimmins). There is a growing body of data on legacy and emerging contaminants in Great Lakes fish tissue. An invaluable data set for trends in legacy chemicals is the longitudinal data from The Great Lakes Fish Monitoring and Surveillance Program (GLFMSP) developed by EPA to evaluate persistent, bioaccumulative and toxic chemical (PBTs) burdens in Great Lakes top predator fish species. There is less information on human burdens of contaminants and even less individual longitudinal human data. Under the auspices of the GLFMSP Clarkson University developed a multipronged approach to detect, confirm and discover “new” PBTs in Great Lakes aquatic biota. In collaboration with Clarkson and the GLFMSP program, DHS will be applying this methodology to serum samples from Great Lakes Basin residents and will compare the human and fish chemical fingerprints. Our lab will take advantage of mass spectrometry-based proteomics to investigate the serum samples to identify the molecular mechanisms that lead to disturbance of the biochemical pathways, based on the level of exposure to legacy contaminants.


Proteogenomics analysis of Lake trout (Salvelinus namaycush). As part of the special studies portion of the Great Lakes Fish Monitoring and surveillance Program (GLFMSP, PI Dr. Thomas Holsen), the proteomic fingerprinting of lake trout is being analyzed. This species is currently used as a bioindicator of chemical stress in the Great Lakes region. Ultimately, these experiments will identify potential protein dysregulations in lake trout that have been exposed to legacy chemical contaminants in the Great Lakes. Since the number of entries for this species’ protein database is small, samples will be searched against databases that will have protein homology to the lake trout. For example, databases of a higher taxonomy to the lake trout, as well as the databases for close relatives (rainbow trout) and distant relatives (zebra fish) will be utilized. The use of these databases will also allow for a glimpse into the homology of proteins among species. De novo sequencing and peptide identification will also be performed. This exploratory research will provide the framework for a direct contaminant/physiological response assessment of top predators furthering the utility of GLFMSP as a monitoring tool for the overall health of the Great Lakes System.


Analysis of transient protein-protein interactions in ephrin signaling. Ephrins and Eph receptors (EphRs) play an important role in nervous system and vascular system development. Disturbances in the ephrin system can lead to nervous system and vascular diseases. Understanding the signal transduction pathways activated by ephrin-EphR interactions is important for studies of both normal development and disease. Stimulation of EphRs leads to activation of signal transduction pathways and the formation of transient protein-protein interactions that trigger cytoskeletal remodeling. However, these protein-protein interactions are not well-studied. We study these intracellular events with a focus on characterizing protein-protein interactions and protein phosphorylation in the neuroblastoma-glyoma cell line as a model system. We have a special interest in investigating the function of a few specific proteins like WAVE1, WAVE2, Trk-fused gene and Nischarin in ephrin signaling.


Studies of Defective Quorum Sensing in Leukemic and Cancer Cells. All cancers contain variable numbers of quiescent cancer stem or proliferative cells that can survive most therapies and which can become reactivated cells and cause recurrence of the clinically evident cancer if the proliferating cancer cells are killed. Quiescent cancer stem cells are usually not killed by drugs designed to kill proliferating cells, and their survival is thus a major reason for failure to cure even highly chemo-sensitive tumors. To understand the abnormalities in quorum sensing that permit cancer stem/progenitor (S/P) cells to far exceed normal homeostatic cell density limits we have been studying a recently obtained p190 bcr-abl driven pre-B cell line (ALL3) derived from the pleural fluid of a patient who was dying of widely disseminated Ph+ ALL resistant to Imatinib and other Bcr-Abl tyrosine kinase inhibitors (TKIs). ALL3 cells, at low densities at which they will not grow spontaneously, can be stimulated to grow by diffusible factors produced by ALL3 cells growing at high density or by cord blood mononuclear (CBM) cells. Our first objective is to identify the proteins or other molecules in the secretomes of the ALL3 cells responsible for the stimulation. Our second objective is to identify the proteins in the secretomes of the CBM cells that are responsible for stimulation of ALL3 cells. To achieve our goals, we perform proteomic analysis of the secreted proteins using biochemical fractionation and nano-liquid chromatography-mass spectrometry (nanoLC-MS/MS), as well as Isotopic Labeling of Amino Acids in Cell Culture (SILAC) in combination with nano-liquid chromatography-mass spectrometry (nanoLC-MS/MS).




Potential P1 peptide (yellow) binding sites on model Hsp90 receptor protein (green).