Quantitative evaluation of peptide-extraction methods by HPLC–triple-quad MS–MS

In this study, the efficiency of five peptide-extraction methods—acetonitrile (ACN) precipitation, ultrafiltration, C18 solid-phase extraction (SPE), dispersed SPE with mesoporous carbon CMK-3, and mesoporous silica MCM-41—was quantitatively investigated. With 28 tryptic peptides as target analytes, these methods were evaluated on the basis of recovery and reproducibility by using high-performance liquid chromatography–triple-quad tandem mass spectrometry in selected-reaction-monitoring mode. Because of the distinct extraction mechanisms of the methods, their preferences for extracting peptides of different properties were revealed to be quite different, usually depending on the pI values or hydrophobicity of peptides. When target peptides were spiked in bovine serum albumin (BSA) solution, the extraction efficiency of all the methods except ACN precipitation changed significantly. The binding of BSA with target peptides and nonspecific adsorption on adsorbents were believed to be the ways through which BSA affected the extraction behavior. When spiked in plasma, the performance of all five methods deteriorated substantially, with the number of peptides having recoveries exceeding 70 % being 15 for ACN precipitation, and none for the other methods. Finally, the methods were evaluated in terms of the number of identified peptides for extraction of endogenous plasma peptides. Only ultrafiltration and CMK-3 dispersed SPE performed differently from the quantitative results with target peptides, and the wider distribution of the properties of endogenous peptides was believed to be the main reason. Distribution of the recoveries of target peptides after treatment by the five peptide-extraction methods