Amino Acid Substitutomics: Profiling Amino Acid Substitutions at the Proteomic Scale Unveils Biological Implications and Escape Hypotheses in Cancer.

Amino acid (AA) substitutions are pivotal modulators of cellular signaling and homeostasis. Current studies of AA substitutions rely mainly on genomic and transcriptomic evidence, while proteome-level characterization remains underexplored, even though such substitutions can arise beyond DNA and RNA mutations. Here we address this gap with PIPI-C, an open-search mass spectrometry tool, and establish an integrated AA substitutomics pipeline to profile AA substitutions across five cancer cohorts and dissect the regulatory roles of substitution-bearing proteins. By cross-referencing 13,399 identified AA substitutions with matched whole-exome sequencing data, we observed no overlap with patient somatic mutations. Specifically, 61% of these substitutions required two or more base alterations and could not be derived from single-nucleotide variants, rendering them exclusively detectable at the proteomic level. The remaining 39% of substitutions were compatible with single-base variations. PIPI-C captured these candidate substitutions at the proteome scale, consistent with a translational-stage origin as reported for alternate RNA decoding in mammals. We further identified notable substitutions, including F43S and E91D in hemoglobin subunit beta, P584T in filamin A, and A175N in fructose-bisphosphate aldolase B, and our pipeline provides a framework for generating hypotheses about AA substitutions in cancer escape.