Identification and Quantification of Murine Mitochondrial Proteoforms Using an Integrated Top-Down and Intact-Mass Strategy

The
development of effective strategies for the comprehensive identification
and quantification of proteoforms in complex systems is a critical
challenge in proteomics. Proteoforms, the specific molecular forms
in which proteins are present in biological systems, are the key effectors
of biological function. Thus, knowledge of proteoform identities and
abundances is essential to unraveling the mechanisms that underlie
protein function. We recently reported a strategy that integrates
conventional top-down mass spectrometry with intact-mass determinations
for enhanced proteoform identifications and the elucidation of proteoform
families and applied it to the analysis of yeast cell lysate. In the
present work, we extend this strategy to enable quantification of
proteoforms, and we examine changes in the abundance of murine mitochondrial
proteoforms upon differentiation of mouse myoblasts to myotubes. The
integrated top-down and intact-mass strategy provided an increase
of ∼37% in the number of identified proteoforms compared to
top-down alone, which is in agreement with our previous work in yeast;
1779 unique proteoforms were identified using the integrated strategy
compared to 1301 using top-down analysis alone. Quantitative comparison
of proteoform differences between the myoblast and myotube cell types
showed 129 observed proteoforms exhibiting statistically significant
abundance changes (fold change >2 and false discovery rate <5%).