鸟枪蛋白质组学
猎枪
计算生物学
肽
基因组
自下而上蛋白质组学
化学
蛋白质组学
氨基酸
肽序列
生物
组合化学
遗传学
生物化学
串联质谱法
基因
质谱法
色谱法
蛋白质质谱法
作者
Taylor J. Lundgren,Patricia L. Clark,Matthew M. Champion
标识
DOI:10.1021/acs.jproteome.3c00730
摘要
Amino acid substitutions (AASs) alter proteins from their genome-expected sequences. Accumulation of substitutions in proteins underlies numerous diseases and antibiotic mechanisms. Accurate global detection of AASs and their frequencies is crucial for understanding these mechanisms. Shotgun proteomics provides an untargeted method for measuring AASs but introduces biases when extrapolating from the genome to identify AASs. To characterize these biases, we created a "ground-truth" approach using the similarities betweenEscherichia coli and Salmonella typhimurium to model the complexity of AAS detection. Shotgun proteomics on mixed lysates generated libraries representing ∼100,000 peptide-spectra and 4161 peptide sequences with a single AAS and defined stoichiometry. Identifying S. typhimurium peptide-spectra with only the E. coli genome resulted in 64.1% correctly identified library peptides. Specific AASs exhibit variable identification efficiencies. There was no inherent bias from the stoichiometry of the substitutions. Short peptides and AASs localized near peptide termini had poor identification efficiency. We identify a new class of "scissor substitutions" that gain or lose protease cleavage sites. Scissor substitutions also had poor identification efficiency. This ground-truth AAS library reveals various sources of bias, which will guide the application of shotgun proteomics to validate AAS hypotheses.
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