To understand and control disease, it is critical to focus on proteins. Proteins consist of their peptide backbone with post-translational modifications (PTMs), the latter of which control the function of the protein. The goal of this research continues to be the development of tools to comprehensively characterize proteins immunoextracted from cells, tissues, or biological matrices at the fmole level. We propose to develop a general platform involving LC/MS and CE-LIF-MS for full glycosylation characterization, including glycan linkage and position isomers at individual sites of multisite glycosylated proteins. All forms on specific sites above the ~10% level will be quantitated. With this information, we will then focus on trace quantitation of multiple forms on multiple sites by multiple reaction monitoring (MRM) LC/MS using our highly sensitive 10 5m i.d. porous layer open tubular (PLOT) LC columns. As a demonstration of the power of the technology, we will immunoprecipitate glycoproteins from small volumes of plasma, and quantitate a number of specific glycoforms on specific sites of healthy individuals and those with breast cancer and other diseases. This technology will allow us to monitor the variation in glycan structures and the relative levels of individual forms on specific sites of glycoproteins for disease vs. control, as well glycan variability within each cohort. A second demonstration will involve full determination of the extracellular domain of the important receptor tyrosine kinases - EGFR and Her2. We will conduct a variety of studies with cell lines for EGFR alone and EGFR/Her2, comprehensively characterizing glycosylation and the dynamics of phosphorylation as a function of stimulation. These results will be compared to those obtained when hypoxia conditions (low oxygen levels) are used for cell growth, as would occur in tissue. Finally, we will demonstrate comprehsive characterization of EGFR extracted from mouse and human tissue. The research will result in powerful new tools to allow a deeper understanding of biological processes and disease.
To understand disease, it is essential that critical target proteins be comprehensively characterized in terms of their peptide backbone structure and post-translational modifications (PTMs). The PTMs control the biological functions of the proteins. We will develop the tools, using LC/MS and CE-LIF-MS, to allow detailed characterization at extent and sensitivity levels not yet possible. Target proteins will be those that are available for therapeutic intervention as well as potential biomarkers of disease. The research will provide powerful tools to help elucidate diseases such as cancer.
|Li, Siyang; Plouffe, Brian D; Belov, Arseniy M et al. (2015) An Integrated Platform for Isolation, Processing, and Mass Spectrometry-based Proteomic Profiling of Rare Cells in Whole Blood. Mol Cell Proteomics 14:1672-83|
|Liu, Zhenke; Dai, Shujia; Bones, Jonathan et al. (2015) A quantitative proteomic analysis of cellular responses to high glucose media in Chinese hamster ovary cells. Biotechnol Prog 31:1026-38|
|Li, Siyang; Nakayama, Tomoko; Akinc, Akin et al. (2015) Development of LC-MS methods for quantitation of hepcidin and demonstration of siRNA-mediated hepcidin suppression in serum. J Pharmacol Toxicol Methods 71:110-9|
|Panikov, Nicolai S; Mandalakis, Manolis; Dai, Shujia et al. (2015) Near-zero growth kinetics of Pseudomonas putida deduced from proteomic analysis. Environ Microbiol 17:215-28|
|Olsen, Lars; Campos, Benito; Winther, Ole et al. (2014) Tumor antigens as proteogenomic biomarkers in invasive ductal carcinomas. BMC Med Genomics 7 Suppl 3:S2|
|Brazin, Kristine N; Mallis, Robert J; Li, Chen et al. (2014) Constitutively oxidized CXXC motifs within the CD3 heterodimeric ectodomains of the T cell receptor complex enforce the conformation of juxtaposed segments. J Biol Chem 289:18880-92|
|Ni, Wenqin; Lin, Melanie; Salinas, Paul et al. (2013) Complete mapping of a cystine knot and nested disulfides of recombinant human arylsulfatase A by multi-enzyme digestion and LC-MS analysis using CID and ETD. J Am Soc Mass Spectrom 24:125-33|
|Ni, Wenqin; Bones, Jonathan; Karger, Barry L (2013) In-depth characterization of N-linked oligosaccharides using fluoride-mediated negative ion microfluidic chip LC-MS. Anal Chem 85:3127-35|
|Laviolette, Laura A; Wilson, Jonas; Koller, Julia et al. (2013) Human folliculin delays cell cycle progression through late S and G2/M-phases: effect of phosphorylation and tumor associated mutations. PLoS One 8:e66775|
|Li, Chen; Rossomando, Anthony; Wu, Shiaw-Lin et al. (2013) Comparability analysis of anti-CD20 commercial (rituximab) and RNAi-mediated fucosylated antibodies by two LC-MS approaches. MAbs 5:565-75|
Showing the most recent 10 out of 44 publications