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.
