Proteomics is an invaluable tool for elucidating the molecular mechanisms that underpin the onset of disease. The modern vision of the proteome encompasses the broad complexity of “protein forms”, or proteoforms, created by specific sets of genetic and chemical modifications that can alter and regulate protein bioactivity. In biomedical research it is pivotal to analyze proteoforms at three distinct levels: first, determination of primary structure, including identity and position of modifications; second, the quantity or abundance of these modifications; and finally, characterization of proteoform assemblies. These protein complexes are held together by weak interactions and are the actual bioactive molecular machines that function in vivo. Currently, proteoforms can only be characterized at the global scale using mass spectrometry (MS). Known as top-down proteomics (TDP), this nascent field has not yet developed into a scalable solution on the level of next generation sequencing or even peptide-based shotgun proteomics, due to the advanced technology required to precisely measure large proteoforms. For this reason, TDP studies are typically limited to proteoforms <30 kDa, which represent less than half of the mammalian proteome. Furthermore, these studies are most often conducted under reducing, denaturing conditions to facilitate preanalytical workflows, so that higher order structure of proteoforms is lost together with cysteine-linked post-translational modifications (PTMs). Fortunately, contemporary structural biologists can analyze multi-proteoform complexes (MPCs) by MS under “native-like” conditions that preserve non-covalent interactions, although this technology is incompatible with high- throughput studies that can be leveraged to discover novel biological insights. Here, we propose a new TDP pipeline that extends the mass range of proteoforms measurable in discovery mode, and also integrates denaturing and native MS to measure MPCs. We will first combine innovative separation techniques and gas-phase chemistries (e.g., novel ion fragmentation techniques and ion-ion reactions) to improve the detection and sequencing of proteoforms up to 100 kDa, and allow the characterization of neglected PTMs. Then, these technologies will be applied to the quantification of proteoforms >30 kDa both in large-scale discovery studies (to identify proteoform expression variations between healthy and disease states) and in targeted experiments (to monitor subsets of proteoforms of interest). Finally, the qualitative and quantitative information collected on single proteoforms through denaturing TDP experiments will be used to facilitate the automatized characterization of MPCs in high-throughput native MS studies. This multi-level, proteoform-centric approach to the global analysis of proteomes will provide researchers with otherwise inaccessible information, opening new possibilities to formulate novel hypotheses for medical treatment and for the discovery of diagnostic disease biomarkers.

Project narrative At the molecular level, the homeostasis of cells and tissues largely depends on the correct activity of properly structured proteins, that if altered can lead to the onset of a myriad different human diseases. With this research program we will develop new mass spectrometry technologies for the analysis of intact protein forms, or proteoforms, and their bioactive complexes, to elucidate the precise set of genetic and chemical modifications responsible for aberrant phenotypes in the human population. These new analytical tools will help advancing our understanding of the mechanisms underlying human pathologies and facilitate the identification of new biomarkers.