Revealing Proteoforms: The Primary Effectors of Biological Function

ABSTRACT The primary focus of my laboratory is the development of new tools and strategies for proteomic analyses of complex biological systems, specifically centered around the concept of the proteoform. Proteoforms, each of which comprises a unique combination of amino acid sequence and post-translational modifications (PTMs), are the primary molecular effectors of cell function. Subtle sequence and PTM differences between proteoforms can completely alter their function and activity. We see comprehensive proteoform-level analysis of biological systems as absolutely essential to understanding their function, for both individual pathways and networks operative within cells, and more globally, to decipher the systems-biology-level dynamics and interactions that control cellular response. The current technology for global proteoform analysis in complex systems is in its infancy, offering both a great challenge and a great opportunity. Our laboratory is keenly interested in tackling this problem and is pioneering a new approach that integrates high resolution proteoform intact mass measurements, both bottom-up and top-down strategies, new informatic tools for the comprehensive analysis of PTMs, and RNA-Seq information; all woven together in a robust bioinformatic framework to allow the comprehensive identification and quantification of proteoforms in complex mixtures. Along with other world-class scientists, we will work towards embarking on the Human Proteoform Project, which includes ambitious subprojects describing the construction and utility of comprehensive proteoform atlases for humans and model organisms. Specific projects in our laboratory will include development of the following: (1) a multi-dimensional separation strategy for increased breadth and depth of proteoform identifications; (2) a source-induced dissociation method for fragmentation of eluting proteoform ions to increase proteoform identifications; (3) intelligent real-time data acquisition; (4) direct acquisition of orbitrap time-domain transients to expand the accessible mass range; (5) data analysis software including the abilities to search for truncated proteoforms and utilize the most abundant mass for identification; (6) sample-specific databases created through integration of bottom-up, top-down, intact mass and RNA-Seq data; (7) visualization tools for manual validation of proteoform identifications and for troubleshooting problems with samples and/or algorithms; and (8) using proteoform quantitative trait loci (QTLs) to reveal the modifying enzymes encoded elsewhere in the genome that are responsible for the critical post-translational modifications with functional consequence. We are excited to develop powerful new tools to advance the state-of-the-art in this new and important field of study to reveal the biologically important effectors of cellular mechanisms. These tools, which will be made widely available to all researchers, will reveal new information essential to the understanding of both normal and disease biology, deepening and accelerating the study of human disease processes.