Widespread noncoded amino acids in human proteome

SUMMARY Proteins are usually deciphered by translation of the coding genome; however, their amino acid residues are seldom determined directly across the proteome. Herein, we describe a systematic workflow for identifying all possible protein residues that differ from the coding genome, termed noncoded amino acids (ncAAs). By measuring the mass differences between the coding amino acids and the actual protein residues in human spermatozoa, over a million nonzero delta masses were detected, fallen into 424 high-quality Gaussian clusters and 571 high-confidence ncAAs spanning 29,053 protein sites. Most ncAAs are novel with unresolved side-chains and discriminative between healthy individuals and patients with oligoasthenospermia. For validation, 40 out of 98 ncAAs that matched with amino acid substitutions were confirmed by exon sequencing. This workflow revealed the widespread existence of previously unreported ncAAs in the sperm proteome, which represents a new dimension on the understanding of amino acid polymorphisms at the proteomic level. Highlights 571 ncAAs spanning 108,000 protein sites were identified in human sperm proteome. Most ncAAs are novel with unresolved sidechains and found at unreported protein sites. Exon sequencing confirmed 40 of 98 ncAAs that matched with amino acid substitutions. Many ncAAs are linked with disease and have potential for diagnosis and targeting. eTOC Blurb We describe a systematic identification of all possible protein residues that were not encoded by their genomic sequences. A total of 571 high-confidence most novel noncoded amino acids were identified in human sperm proteome, corresponding to over 108,000 ncAA-containing protein sites. For validation, 40 out of 98 ncAAs that matched to amino acid substitutions were confirmed by exon sequencing. These ncAAs are discriminative between individuals and expand our understanding of amino acid polymorphisms in human proteomes and diseases.