Absolute Quantitation of Low Abundance Plasma APL1β peptides at Sub-fmol/mL Level by SRM/MRM without Immunoaffinity Enrichment

• Published in: Journal of proteome research 2014-02, Vol.13 (2), p.1012-1020
• Main Authors: Sano, Shozo, Tagami, Shinji, Hashimoto, Yuuki, Yoshizawa-Kumagaye, Kumiko, Tsunemi, Masahiko, Okochi, Masayasu, Tomonaga, Takeshi
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Chaperonin 60 - blood; Chaperonin 60 - chemistry; Chromatography, Affinity - methods; Electrophoresis, Polyacrylamide Gel; Humans; Limit of Detection; Molecular Sequence Data; Peptide Fragments - blood; Peptide Fragments - chemistry; Reference Standards
• ISSN: 1535-3893; 1535-3907
• DOI: 10.1021/pr4010103

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 Aβ-like peptides (APL1β), 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 C18-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 APL1β 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 APL1β 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.