Absolute Quantitation of Low Abundance Plasma APL1 beta peptides at Sub-fmol/mL Level by SRM/MRM without Immunoaffinity Enrichment

作者：Sano Shozo; Tagami Shinji; Hashimoto Yuuki; Yoshizawa Kumagaye Kumiko; Tsunemi Masahiko; Okochi Masayasu; Tomonagat Takeshi^*

来源：Journal of Proteome Research, 2014, 13(2): 1012-1020.

DOI：10.1021/pr4010103

• SRM/MRM low abundant plasma peptide
• peptide enrichment
• Alzheitner's disease

Selected/multiple reaction monitoring (SRM/MRM) has been widely used for the quantification of specific proteins/peptides, although it is still challenging to quantitate low abundant proteins/peptides in complex samples such as plasma/serum. To overcome this problem, enrichment of target proteins/peptides is needed, such as immunoprecipitation; however, this is labor-intense and generation of antibodies is highly expensive. In this study, we attempted to quantify plasma low abundant APLP1-derived A beta-like peptides (APL1 beta), a surrogate marker for Alzheimer's disease, by SRM/MRM using stable isotope-labeled reference peptides without immunoaffinity enrichment. A combination of Cibacron Blue dye mediated albumin removal and acetonitrile extraction followed by C-18-strong cation exchange multi-StageTip purification was used to deplete plasma proteins and unnecessary peptides. Optimal and validated precursor ions to fragment ion transitions of APL1 beta were developed on a triple quadruple mass spectrometer, and the nanoliquid chromatography gradient for peptide separation was optimized to minimize the biological interference of plasma. Using the stable isotope-labeled (SI) peptide as an internal control, absolute concentrations of plasma APL1 beta peptide could be quantified as several hundred amol/mL. To our knowledge, this is the lowest detection level of endogenous plasma peptide quantified by SRM/MRM.

• 出版日期2014-2