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, 2) Jumping Translocation Breakpoint (JTB) proteins and 3) Biomarker discovery for Breast Cancer using human breast milk and serum. Many other proteomics projects are either underway or just started such as the biochemical events which occur due to environmental exposure, persistent, bio-accumulative and toxic (PBTs) chemical in the Great Lakes Regions and their effects on top predator species of fish and humans in this region. Some of these projects include:

Proteomics analysis of human breast milk in women with breast cancer and matched controls. Breast cancer (BC) arises from cells with genetic and "epigenetic" changes. The epigenetic, or non-sequence 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 T. 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.
 Initially, preliminary milk studies were performed to determine if proteins were dysregulated in the breast milk of women with BC and without. These studies included both across women comparisons (BC milk from one donor and control milk from a separate donor) and within woman comparisons (BC milk is from the diseased breast and control milk is from the other breast). Both gel-based and in-solution based proteomic methods were used to determine proteins which were dysregulated between the groups. Proteins which were found to be a consistently dysregulated include actin family proteins, fatty acid binding proteins, lactoferrin, lysozyme, perilipin, titin family proteins, xanthine dehydrogenase/oxidase and zinc-alpha-2 glycoprotein. These proteins identified are currently being validated using a larger set of breast milk. Additionally, lactoferrin is the most abundant protein in breast milk and its presence can interfere with lower abundant proteins being identified and analyzed. Therefore, antibodies against lactoferrin were made and we are currently in the process of depleting breast milk samples of lactoferrin to allow us to identify lower abundant proteins. The new milk samples, once depleted will be run using in-gel and in-solution-based proteomics methods.
 To move forward from the preliminary milk studies, Drs. Pentecost and Darie are working with human serum to identify proteins which were previously found in breast milk and any additional proteins unique to sera. The serum studies include larger sample groups (48 vs 48) as well as smaller comparison groups which look at specific breast cancer types (stage IIA, IIB etc.) or specific subtypes of BC (hormone positive, triple negative etc.). Here we aim to achieve a draft biomarker set for serum which will be further validated in future targeted experiments. The outcomes of the proteomics experiments for both breast milk and serum will be analyzed using bioinformatics softwares such as gene set enrichment analysis (GSEA), Ingenuity Pathway Analysis, STRING analysis, Reactome etc. Any proteins of particular interest from both the milk and serum studies will be further analyzed using targeted quantitative proteomics. Absolute quantification (AQUA) or multiple reaction monitoring (MRM) based experiments will be employed to further quantify the proteins of interest from the untargeted experiments.
 The idea is to determine a protein biomarker set in which women of any age can be screened for breast cancer. The team from Clarkson University is responsible for biochemical fractionation and mass spectrometric identification of proteins from the human breast milk and serum, 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 and serum samples from healthy women versus women with breast cancer. The overarching goal is to identify proteins which are dysregulated between control and cancer samples which can lead to a biomarker for breast cancer.

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 downregulated plasmid due to the JTB protein having a function in mitosis. The JTB downregulated plasmid contains His and Flag tags and shRNA. 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 G2 and Xevo G2-XS 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.

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.

Developing a method to monitor estrogen-inducible proteins in fishes from the Great Lakes. Additionally, the expression of liver proteins in lake trout (Salvelinus namaycush) and walleye (Sander vitreus) are being monitored for their exposure levels to environmental contaminants (within the Great Lakes). Some of these contaminants, like polychlorinated biphenyls (PCBs), have estrogenic activity (estrogenic disrupting compounds (EDCs)). Two important fish proteins whose levels change upon exposure to EDCs are vitellogenin and zona radiate proteins (aka zona pellucida or vitelline envelope proteins). These are prime candidates for this study for several reasons: a) the levels of these proteins are increased upon exposure to EDCs, b) both proteins are produced in male fish only upon exposure to EDCs, c) both proteins are only produced in the liver.

Proteomics-based analysis of proteins. We have used mass spectrometry-based proteomics to identify oxidative stress in proteins. This is done by identification of 4-hydroxy-2-nonenal (HNE) adducts on amino acids. HNE modifications usually occur of cysteine, histidine and/or lysine residues which results in a mass increase. We are using MS-based techniques to identify the MS signatures of amino acids with HNE modification using two model proteins, lysozyme and bovine serum albumin (BSA). Comparison of spectra between unmodified and HNE-modified proteins gives insight on how HNE is affecting the protein, and how many HNE modifications can occur per one lysozyme or BSA molecule. A second project we are currently working on is optimization of the gel-based proteomics experiment. The current process is time consuming, and determining an acceptable and quicker method can be useful. Here we are also using two model proteins, lysozyme and BSA as the molecular weight and number of disulfide bonds is well known. To do this, we are changing the digestion time and temperature and also different variations of extraction with both shaking and sonication (i.e. number of steps, time at each step etc.). All of these are compared to a normal gel-based experiment, and we are looking for acceptable or better protein identification scores compared to the control set to determine whether a method is comparable.

Proteomic analysis of lymphoblastoid cell lines from patients with amyotrophic lateral sclerosis (ALS). This project is a collaboration with Dr. Aurelian Radu at Ichan School of Medicine at Mount Sinai. We are looking into how motor neurons are degenerating as ALS progresses. It is difficult to study the pathological processed in live patients as these motor neurons are part of the motor cortex, brain stem nuclei and anterior horn of the spinal cord. To combat this problem, a model cell line to these motor neurons; lymphoblasoid cell lines. From initial experiments, we have found proteins which are known to be involved in ALS progression, while also identifying additional proteins which are of interest for future proteomic investigations. Currently we have one paper published, and are looking to expand this study.

Past Projects

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).

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).