RESUMO
In contrast to quantification of biotherapeutics, endogenous protein biomarker and target quantification using LC-MS based targeted proteomics can require a much more stringent and time-consuming tryptic signature peptide selection for each specific application. While some general criteria exist, there are no tools currently available in the public domain to predict the ionization efficiency for a given signature peptide candidate. Lack of knowledge of the ionization efficiencies forces investigators to choose peptides blindly, thus hindering method development for low abundant protein quantification. Here, the authors propose a tryptic signature peptide selection workflow to achieve a more efficient method development and to improve success rates in signature peptide selection for low abundant endogenous target and protein biomarker quantification.
Assuntos
Proteômica , Espectrometria de Massas em Tandem , Cromatografia Líquida , Fluxo de Trabalho , Peptídeos , BiomarcadoresRESUMO
Background: Currently, no regulatory guidelines are available for parallelism assessment for LC-MS biomarker quantification. Spike recovery, standard addition and dilutional linearity are recommended with no mention of the implications of applying these approaches. Results: Here, using human urine creatinine, the authors compared spike recovery and standard addition in LC-MS biomarker quantification, and evaluated a new hybrid approach: parallelism QCs. The authors drew different conclusions based on which approach was used (<15% cutoff). Conclusion: Current recommended approaches may lead to different conclusions and are not equivalent and interchangeable. The authors recommend that standard addition should be the universal 'go-to' method for LC-MS biomarker parallelism assessment; parallelism QCs, which consider the total concentration as the theoretical value, can be used if the authentic matrix is limited.
