My research focuses on the study of the proteins present in an organism, or proteomics, using high resolution mass spectrometry. Traditionally, proteomics is carried out by enzymatically digesting the original proteins into short peptides, which are easier to analyze – a process similar to the “shotgun” approach normally used in genomics. This methodology, known as bottom-up proteomics, fails to reconstruct the original complexity of a proteome, as the correlation between different sources of variation – e.g., genetic, such as single nucleotide polymorphisms inducing a single amino acid change, or chemical, such as post-translational modifications – within a single gene product is lost and cannot be inferred starting from proteolytic peptides. Therefore, my research group will apply instead top-down proteomics, a novel approach based on the analysis of intact, undigested protein forms known as proteoforms. We will use top-down proteomics to study the specific modification patterns of proteoforms differentially localized in various sub-cellular compartments, or how different proteoforms derived from the same gene can distinguish between healthy and aberrant phenotypes.

Research projects where top-down proteomics is applied to biology and translational sciences:

• Cancer biology: quantitative analysis of proteoforms distinguishing healthy tissues vs tumors, with specific focus on key oncogenes and their modifications, which have important implications in the onset and progression of disease;
• Development of a systems biology platform centered on the concept of proteoform to investigate biological processes at the molecular level in model organisms (e.g., budding yeast);
• Improvement of mass spectrometry-based solutions for the characterization of large proteins and protein complexes: the proteomes of mammals consist primarily of proteins with molecular weight between 30 and 60 kDa, and often their biological functions are carried out in multi-proteoforms complexes whose stoichiometry and bioactivity can be modulated by post-translational modifications and binding with small molecules (ligands); hence the importance of mass spectrometry tools specifically designed to analyze large biomolecules.

(full list of publications here: https://scholar.google.com/citations?user=e3qaG7UAAAAJ&hl=en)

• Ntai I.*, Fornelli L.*, DeHart C.J.*, Hutton J.E., Doubleday P.F., LeDuc R.D., van Nispen A.J., Fellers R.T., Whiteley G., Boja E.S., Rodriguez H., Kelleher N.L. (2018) Precise characterization of KRAS4b proteoforms in human colorectal cells and tumors reveals mutation/modification cross-talk. PNAS, doi: 10.1073/pnas.1716122115
• Skinner O.S., Haverland N.A., Fornelli L., Melani R.D., Do Vale L.H.F., Seckler H.S., Doubleday P.F., Schachner L.F., Srzentić K., Kelleher N.L., Compton P.D. (2018) Top-down characterization of endogenous protein complexes with native proteomics. Nat Chem Biol, doi: 10.1038/nchembio.2515
• Fornelli L., Durbin K.R., Fellers R.T., Early B.P., Greer J.B., LeDuc R.D., Compton P.D., Kelleher N.L. (2017) Advancing Top-down Analysis of the Human Proteome Using a Benchtop Quadrupole-Orbitrap Mass Spectrometer. J Proteome Res., doi: 10.1021/acs.jproteome.6b00698
• Fornelli L., Ayoub D., Aizikov K., Liu X., Damoc E., Pevzner P.A., Makarov A., Beck A., Tsybin Y.O. (2017) Top-down analysis of immunoglobulin G isotypes 1 and 2 with electron transfer dissociation on a high-field Orbitrap mass spectrometer. J Proteomics, doi: 10.1016/j.jprot.2017.02.013
• Zheng Y.*, Fornelli L.*, Compton P.D., Sharma S., Canterbury J., Mullen C., Zabrouskov V., Fellers R.T., Thomas P.M., Licht J.D., Senko M.W., Kelleher N.L. (2016) Unabridged Analysis of Human Histone H3 by Differential Top-Down Mass Spectrometry Reveals Hypermethylated Proteoforms from MMSET/NSD2 Overexpression. Mol Cell Proteomics, doi: 10.1074/mcp.M115.053819
• 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., 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. (2016) An informatic framework for decoding protein complexes by top-down mass spectrometry. Nat Methods, doi: 10.1038/nmeth.3731
• Fornelli L.*, Ayoub D.*, Aizikov K., Beck A., Tsybin Y.O. (2014) Middle-down analysis of monoclonal antibodies with electron transfer dissociation Orbitrap Fourier transform mass spectrometry. Anal Chem, doi: 10.1021/ac4036857
• Fornelli L., Damoc E., Thomas P.M., Kelleher N.L., Aizikov K., Denisov E., Makarov A., Tsybin Y.O. (2012) Analysis of intact monoclonal antibody IgG1 by electron transfer dissociation Orbitrap FTMS. Mol Cell Proteomics, doi: 10.1074/mcp.M112.019620