Intact-Mass Analysis Facilitating the Identification of Large Human Heart Proteoforms

Proteoforms, the
primary effectors of biological processes, are
the different forms of proteins that arise from molecular processing
events such as alternative splicing and post-translational modifications.
Heart diseases exhibit changes in proteoform levels, motivating the
development of a deeper understanding of the heart proteoform landscape.
Our recently developed two-dimensional top-down proteomics platform
coupling serial size exclusion chromatography (sSEC) to reversed-phase
chromatography (RPC) expanded coverage of the human heart proteome
and allowed observation of high-molecular weight proteoforms. However,
most of these observed proteoforms were not identified due to the
difficulty in obtaining quality tandem mass spectrometry (MS2) fragmentation
data for large proteoforms from complex biological mixtures on a chromatographic
time scale. Herein, we sought to identify human heart proteoforms
in this data set using an enhanced version of Proteoform Suite, which
identifies proteoforms by intact mass alone. Specifically, we added
a new feature to Proteoform Suite to determine candidate identifications
for isotopically unresolved proteoforms larger than 50 kDa, enabling
subsequent MS2 identification of important high-molecular weight human
heart proteoforms such as lamin A (72 kDa) and trifunctional enzyme
subunit α (79 kDa). With this new workflow for large proteoform
identification, endogenous human cardiac myosin binding protein C
(140 kDa) was identified for the first time. This study demonstrates
the integration of our sSEC-RPC-MS proteomics platform with intact-mass
analysis through Proteoform Suite to create a catalog of human heart
proteoforms and facilitate the identification of large proteoforms
in complex systems.