Neil Kelleher (Presenter)
Bio: Neil L. Kelleher, PhD Walter & Mary Elizabeth Glass Chair in the Life Sciences; Professor of Molecular Biosciences and of Chemistry, Weinberg College of Arts and Sciences Professor of Medicine, Feinberg School of Medicine Director, Proteomics Center of Excellence With more than 300 papers published over the course of his career and teaching duties in two departments, Dr. Kelleher is a trans-disciplinary investigator with international impact in mass spectrometry-based proteomics and the discovery of new natural products from the microbial world. The Kelleher group has successfully driven both technology development and applications of high performance mass spectrometry at the interface of chemistry and biology. Dr. Kelleher’s laboratory comprises three main sub-groups working in the areas of Top Down Proteomics, Biosynthesis/Discovery of Natural Products, and Chromatin Biology.
Authorship: The Kelleher Research Group
A general theme in translational proteomics that involves first genotyping patients will be presented. Some peptide-driven assays for histone modifications in cancer epigenetics will be presented, along with whole-protein (i.e., top down proteomic) measurements of high value targets like KRAS. Assays deployed in clinical research are typically developed and validated with basic studies ofisogenic cell-based models of colorectal cancer, multiple myeloma, and glioblastoma. Current state-of-the-art in whole protein mass spectrometry will be presented, along with the state of play in assessing the value of proteoform-resolved measurements in clinical research and biomarker discovery & verification.
Top Down and Bottom Up Proteomics: A Brief Primer
After some years spent increasing metrics of proteome coverage, the field of proteomics has increasingly focused on the quality of information generated during interrogation of living systems. Another aspect trending presently is to integrate proteomics with data from other “-omics” in order to gain deeper insights into cellular and disease biology. From this perspective, it is apparent that the analysis of intact proteoforms, or top-down proteomics (TDP),1 presents additional advantages. In providing precise compositional information, TDP can add molecular details lost when proteoforms are dissected into proteolytic peptides used in bottom-up proteomics (BUP). Although BUP can identify and localize post-translational modifications (PTMs) on proteins, the well-known ‘protein inference problem’ greatly complicates the elucidation of their global patterns or cross-talk, aspects which can be captured using TDP. This is exemplified by the so-called “histone code”, where PTMs comprise combinatorial and highly dynamic patterns resulting from concerted interactions between prior PTMs and histone-modifying enzymes. These patterns govern the reading of histone marks and myriad biomolecular activities, and can be comprehensively described at the proteoform level to help assign the functions of PTM patterns.
It is true that TDP is technologically challenging, yet perceptions about this are often historical and not updated quickly in the minds of experts or those far afield. Rapid advances in instrumentation by most manufacturers over the decade have rendered targeted and high-throughput TDP feasible—even on the benchtop mass spectrometers of today. Moreover, a collection of TDP practitioners have founded the Consortium for Top Down Proteomics (CTDP, http://topdownproteomics.org/), many of which have contributed articles to this special issue. CTDP has the aims of accelerating impactful and collaborative research,2 increasing the visibility of TDP within the community, and promulgating knowledge and best practices for newcomers to the field. Notably, the first CTDP manuscript rallied interest and focus around the “proteoform” and now has over 220 citations.3 As further recognition of the accelerating progress in TDP, the UniProt Knowledgebase has begun cross-referencing their accession numbers with the permanent PFR identifiers in the proteoform atlas hosted by the CTDP (http://repository.topdownproteomics.org/).
Denaturing and Native Top-Down MS
Typically, TDP has been performed under denaturing conditions, following workflows originally developed for BUP. Namely, proteins are isolated from cells in detergent-containing buffers, and subjected to pre-fractionation to reduce individual sample complexity. In recent years, combined with concurrent advances in dedicated high-throughput data analysis platforms, TDP has been sufficiently optimized to enable a degree of qualitative and quantitative proteome coverage more typically achieved by BUP. Today, high-confidence identification and characterization of several thousand proteoforms is feasible.4-6 However, the number of proteoforms identified by denaturing TDP drops off at molecular weights (MW) exceeding ~50-70 kDa due to technical issues associated with both the limitations of mass spectrometry and the need for improved separations.
Another option for high mass proteins derives from native top-down mass spectrometry (nTDMS), which preserves non-covalent interactions, labile PTMs, cofactors and physiological stoichiometry.7 Native TD provides intact mass spectra characterized by fewer and lower charge states appearing at higher m/z values. Although nTDMS has traditionally been employed for the targeted analysis of purified proteins, the high-MW capabilities of nTDMS can be employed on-line when combined with the appropriate protein separation techniques such as ion exchange chromatography.8 With many analytical benefits and increased accessibility to biologically relevant protein structures and interactions, nTDMS has the potential to not only access the higher-MW regions of the human proteome but also to provide enhanced information and molecular detail.
When will a tipping point be reached?
As it stands today, BUP and TDP stand as highly complementary in terms of value proposition to both researchers and stakeholders. While BUP remains unmatched in depth of proteome coverage and quantitation of peptides, TDP characterizes proteoforms and their variants directly. Native TD can hone in on endogenous protein complexes.9 As techniques for TDP and nTDMS continue to evolve, one could extrapolate toward a tipping point where the analysis of intact proteins becomes far more widespread. The “Why?” for this change is becoming more clear — great progress in proteoform-resolved measurements has the value of high molecular specificity when it comes to protein molecules, often with strong mechanistic connections to pathology. As an example, a recent report of phosphorylated alpha-synuclein proteoforms in Parkinson’s Disease10 demonstrates the new kind of information being provided by proteoform-resolved measurements. The “When?” is the crux of the issue: when will the value of these measurements lead to wider adoption and deployment of the technology in academic and clinical research? There is a common assertion that TDP is several years behind BUP in terms of acceptance and implementation in the field. If true, then perhaps increasing numbers of practitioners will adopt the direct interrogation of intact proteins and their protein complexes. Advancements in TDP like those enclosed in this special issue will be the driving agents of that change, and the implications for the study of protein molecules and the translation of this knowledge to understanding disease hold a good deal of unrealized potential.
References & Acknowledgements:
(1) Toby, T. K.; Fornelli, L.; Kelleher, N. L. Progress in Top-Down Proteomics and the Analysis of Proteoforms. Annu Rev Anal Chem (Palo Alto Calif) 2016, 9, 499-519.
(2) Dang, X.; Scotcher, J.; Wu, S.; Chu, R. K.; Tolic, N.; Ntai, I.; Thomas, P. M.; Fellers, R. T.; Early, B. P.; Zheng, Y.; Durbin, K. R.; Leduc, R. D.; Wolff, J. J.; Thompson, C. J.; Pan, J.; Han, J.; Shaw, J. B.; Salisbury, J. P.; Easterling, M.; Borchers, C. H.; Brodbelt, J. S.; Agar, J. N.; Pasa-Tolic, L.; Kelleher, N. L.; Young, N. L. The first pilot project of the consortium for top-down proteomics: a status report. Proteomics 2014, 14, 1130-1140.
(3) Smith, L. M.; Kelleher, N. L.; Consortium for Top Down, P. Proteoform: a single term describing protein complexity. Nat Methods 2013, 10, 186-187.
(4) Durbin, K. R.; Fornelli, L.; Fellers, R. T.; Doubleday, P. F.; Narita, M.; Kelleher, N. L. Quantitation and Identification of Thousands of Human Proteoforms below 30 kDa. J Proteome Res 2016, 15, 976-982.
(5) Anderson, J. C.; Wan, Y.; Kim, Y. M.; Pasa-Tolic, L.; Metz, T. O.; Peck, S. C. Decreased abundance of type III secretion system-inducing signals in Arabidopsis mkp1 enhances resistance against Pseudomonas syringae. Proc Natl Acad Sci U S A 2014, 111, 6846-6851.
(6) Vorontsov, E. A.; Rensen, E.; Prangishvili, D.; Krupovic, M.; Chamot-Rooke, J. Abundant Lysine Methylation and N-Terminal Acetylation in Sulfolobus islandicus Revealed by Bottom-Up and Top-Down Proteomics. Mol Cell Proteomics 2016, 15, 3388-3404.
(7) Skinner, O. S.; Havugimana, P. C.; Haverland, N. A.; Fornelli, L.; Early, B. P.; Greer, J. B.; Fellers, R. T.; Durbin, K. R.; Do Vale, L. H. F.; Melani, R. D.; Seckler, H. S.; Nelp, M. T.; Belov, M. E.; Horning, S. R.; Makarov, A. A.; LeDuc, R. D.; Bandarian, V.; Compton, P. D.; Kelleher, N. L. An informatic framework for decoding protein complexes by top-down mass spectrometry. Nat Methods 2016, 13, 237-240.
(8) Muneeruddin, K.; Nazzaro, M.; Kaltashov, I. A. Characterization of Intact Protein Conjugates and Biopharmaceuticals Using Ion-Exchange Chromatography with Online Detection by Native Electrospray Ionization Mass Spectrometry and Top-Down Tandem Mass Spectrometry. Anal Chem 2015, 87, 10138-10145.
(9) Ntai, I.; LeDuc, R. D.; Fellers, R. T.; Erdmann-Gilmore, P.; Davies, S. R.; Rumsey, J.; Early, B. P.; Thomas, P. M.; Li, S.; Compton, P. D.; Ellis, M. J.; Ruggles, K. V.; Fenyo, D.; Boja, E. S.; Rodriguez, H.; Townsend, R. R.; Kelleher, N. L. Integrated Bottom-Up and Top-Down Proteomics of Patient-Derived Breast Tumor Xenografts. Mol Cell Proteomics 2016, 15, 45-56.
(10) Kellie, J. F.; Higgs, R. E.; Ryder, J. W.; Major, A.; Beach, T. G.; Adler, C. H.; Merchant, K.; Knierman, M. D. Quantitative measurement of intact alpha-synuclein proteoforms from post-mortem control and Parkinson's disease brain tissue by intact protein mass spectrometry. Sci Rep 2014, 4, 5797.
The authors would like to acknowledge the W.M. Keck foundation for generous support and funding (DT061512). In addition, this work was partially supported the National Resource for Translational and Developmental Proteomics under Grant P41 GM108569 from the National Institute of General Medical Sciences, National Institutes of Health.
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