Quantifying homologous proteins and proteoforms

Many proteoforms – arising from alternative splicing, post-translational modifications (PTMs), or paralogous genes – have distinct biological functions, such as histone PTM proteoforms. However, their quantification by existing bottom-up mass–spectrometry (MS) methods is undermined by peptide-specific biases. To avoid these biases, we developed and implemented a first-principles model (HI quant ) for quantifying proteoform stoichiometries. We characterized when MS data allow inferring proteoform stoichiometries by HI quant , derived an algorithm for optimal inference, and demonstrated experimentally high accuracy in quantifying fractional PTM occupancy without using external standards, even in the challenging case of the histone modification code. A HI quant server is implemented at: https://web.northeastern.edu/slavov/2014_HIquant/