Elucidating Proteoform Families from Proteoform Intact-Mass and Lysine-Count Measurements

Proteomics
is presently dominated by the “bottom-up”
strategy, in which proteins are enzymatically digested into peptides
for mass spectrometric identification. Although this approach is highly
effective at identifying large numbers of proteins present in complex
samples, the digestion into peptides renders it impossible to identify
the proteoforms from which they were derived. We present here a powerful
new strategy for the identification of proteoforms and the elucidation
of proteoform families (groups of related proteoforms) from the experimental
determination of the accurate proteoform mass and number of lysine
residues contained. Accurate proteoform masses are determined by standard
LC–MS analysis of undigested protein mixtures in an Orbitrap
mass spectrometer, and the lysine count is determined using the NeuCode
isotopic tagging method. We demonstrate the approach in analysis of
the yeast proteome, revealing 8637 unique proteoforms and 1178 proteoform
families. The elucidation of proteoforms and proteoform families afforded
here provides an unprecedented new perspective upon proteome complexity
and dynamics.