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)
Project #
Application #
Study Section
Enabling Bioanalytical and Imaging Technologies Study Section (EBIT)
Program Officer
Edmonds, Charles G
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Northwestern University at Chicago
Schools of Arts and Sciences
United States
Zip Code
Swaroop, Alok; Oyer, Jon A; Will, Christine M et al. (2018) An activating mutation of the NSD2 histone methyltransferase drives oncogenic reprogramming in acute lymphocytic leukemia. Oncogene :
Fornelli, Luca; Srzenti?, Kristina; Huguet, Romain et al. (2018) Accurate Sequence Analysis of a Monoclonal Antibody by Top-Down and Middle-Down Orbitrap Mass Spectrometry Applying Multiple Ion Activation Techniques. Anal Chem 90:8421-8429
Fornelli, Luca; Durbin, Kenneth R; Fellers, Ryan T et al. (2017) Advancing Top-down Analysis of the Human Proteome Using a Benchtop Quadrupole-Orbitrap Mass Spectrometer. J Proteome Res 16:609-618
Haverland, Nicole A; Skinner, Owen S; Fellers, Ryan T et al. (2017) Defining Gas-Phase Fragmentation Propensities of Intact Proteins During Native Top-Down Mass Spectrometry. J Am Soc Mass Spectrom 28:1203-1215
Hattori, Takamitsu; Lai, Darson; Dementieva, Irina S et al. (2016) Antigen clasping by two antigen-binding sites of an exceptionally specific antibody for histone methylation. Proc Natl Acad Sci U S A 113:2092-7
Zheng, Yupeng; Fornelli, Luca; Compton, Philip D et al. (2016) Unabridged Analysis of Human Histone H3 by Differential Top-Down Mass Spectrometry Reveals Hypermethylated Proteoforms from MMSET/NSD2 Overexpression. Mol Cell Proteomics 15:776-90
Savaryn, John Paul; Skinner, Owen S; Fornelli, Luca et al. (2016) Targeted analysis of recombinant NF kappa B (RelA/p65) by denaturing and native top down mass spectrometry. J Proteomics 134:76-84
Toby, Timothy K; Fornelli, Luca; Kelleher, Neil L (2016) Progress in Top-Down Proteomics and the Analysis of Proteoforms. Annu Rev Anal Chem (Palo Alto Calif) 9:499-519
Durbin, Kenneth R; Fornelli, Luca; Fellers, Ryan T et al. (2016) Quantitation and Identification of Thousands of Human Proteoforms below 30 kDa. J Proteome Res 15:976-82
Skinner, Owen S; Havugimana, Pierre C; Haverland, Nicole A et al. (2016) An informatic framework for decoding protein complexes by top-down mass spectrometry. Nat Methods 13:237-40

Showing the most recent 10 out of 103 publications