The division of protein mass spectrometry into the dichotomy of Top Down and Bottom Up has emerged over the past decade. For many of these years Top Down has been seen as a specialized form of mass spectrometry (MS) that is ill-suited for high throughput analysis. The content of this A1-submission for renewal will help to change this perception. All major aspects of Top Down have improved over the last four years, including;front end separations, instrumentation and data analysis. Recent advances have allowed us to detect 1000 proteins and over 4000 isoforms from ~20 milion human cancer cells in 8 runs over a period of a few months. We propose further innovations to refine a sample preparation platform that has evolved from a low-resolution 2-dimensional separation into a 3-dimensional approach over the last funding period. We now propose a combination of solution isoelectric focusing (sIEF) and gel eluted liquid fraction entrapment electrophoresis (GELFrEE) prior to nanocapillary LC-MS/MS using a next-generation Orbitrap (Aim #1). Based on preliminary data obtained through an ongoing collaboration with the group of Alexander Makarov in Bremen, we project a major expansion in the number of labs capable of performing Top Down Mass Spectrometry on complex mixtures. We will continue with an "engineering ethos" for technology development to define the Human Proteome, and provide ample preliminary data where proteins with multiple PTMs are found to change in cancer cells undergoing the phenomenon of senescence (Aim #2). Unlike apoptosis, senescence occurs when cells irreversibly stop dividing but remain alive. In comparison to apoptosis, the study of senescence is a relatively new field that has become popular in recent years for its strong connection to aging and its potential role in the treatment of cancer. In response to prior review, we have prepared a new Aim 3 and combined our work on cellular-senescence with an expanded set of preliminary data on integral membrane proteins from mitochondria with up to 12 transmembrane domains. Overall, we describe a maturation of the technology where observations made using Top Down (such as the strong up regulation of methylations on a HMG type protein in senescence) can generate hypothesis-driven work on 'high value'observations. Given the growing interest in Top Down Mass Spectrometry, we project major advances over the next granting period for discovery type biological and biomedical research using whole protein mass spectrometry.

Public Health Relevance

This project entails continued development of Top Down Mass Spectrometry into a proteomic technology that provides a clarified picture of intracellular signaling events at the level of protein molecules. The proposed granting period would extend exemplars in human proteomics to accelerate the adoption of the Top Down philosophy of protein analysis as an enabling approach in hundreds and eventually thousands of laboratories worldwide.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Enabling Bioanalytical and Imaging Technologies Study Section (EBIT)
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Edmonds, Charles G
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Northwestern University at Chicago
Schools of Arts and Sciences
United States
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Ye, Hui; Mandal, Rakesh; Catherman, Adam et al. (2014) Top-down proteomics with mass spectrometry imaging: a pilot study towards discovery of biomarkers for neurodevelopmental disorders. PLoS One 9:e92831
Skinner, Owen S; Catherman, Adam D; Early, Bryan P et al. (2014) Fragmentation of integral membrane proteins in the gas phase. Anal Chem 86:4627-34
Hong, Seok Hoon; Ntai, Ioanna; Haimovich, Adrian D et al. (2014) Cell-free protein synthesis from a release factor 1 deficient Escherichia coli activates efficient and multiple site-specific nonstandard amino acid incorporation. ACS Synth Biol 3:398-409
Durbin, Kenneth R; Fellers, Ryan T; Ntai, Ioanna et al. (2014) Autopilot: an online data acquisition control system for the enhanced high-throughput characterization of intact proteins. Anal Chem 86:1485-92
LeDuc, Richard D; Fellers, Ryan T; Early, Bryan P et al. (2014) The C-score: a Bayesian framework to sharply improve proteoform scoring in high-throughput top down proteomics. J Proteome Res 13:3231-40
Zheng, Yupeng; Tipton, Jeremiah D; Thomas, Paul M et al. (2014) Site-specific human histone H3 methylation stability: fast K4me3 turnover. Proteomics 14:2190-9
Cannon, Joe R; Cammarata, Michael B; Robotham, Scott A et al. (2014) Ultraviolet photodissociation for characterization of whole proteins on a chromatographic time scale. Anal Chem 86:2185-92
Li, Yihan; Compton, Philip D; Tran, John C et al. (2014) Optimizing capillary electrophoresis for top-down proteomics of 30-80 kDa proteins. Proteomics 14:1158-64
Dang, Xibei; Scotcher, Jenna; Wu, Si et al. (2014) The first pilot project of the consortium for top-down proteomics: a status report. Proteomics 14:1130-40
Popovic, Relja; Martinez-Garcia, Eva; Giannopoulou, Eugenia G et al. (2014) Histone methyltransferase MMSET/NSD2 alters EZH2 binding and reprograms the myeloma epigenome through global and focal changes in H3K36 and H3K27 methylation. PLoS Genet 10:e1004566

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