title: Absolute quantitation of low abundance plasma APL1β peptides at sub-fmol/mL Level by SRM/MRM without immunoaffinity enrichment.
abstract: 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.
authors.affiliation: Laboratory of Proteome Research, National Institute of Biomedical Innovation , Osaka 567-0085, Japan.
authors.forename: Shozo
authors.initials: S
authors.lastname: Sano
authors.forename: Shinji
authors.initials: S
authors.lastname: Tagami
authors.forename: Yuuki
authors.initials: Y
authors.lastname: Hashimoto
authors.forename: Kumiko
authors.initials: K
authors.lastname: Yoshizawa-Kumagaye
authors.forename: Masahiko
authors.initials: M
authors.lastname: Tsunemi
authors.forename: Masayasu
authors.initials: M
authors.lastname: Okochi
authors.forename: Takeshi
authors.initials: T
authors.lastname: Tomonaga
mesh_terms: D000595:Amino Acid Sequence; D018834:Chaperonin 60 / Q000097:blood* / Q000737:chemistry; D002846:Chromatography, Affinity / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D057230:Limit of Detection; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000097:blood* / Q000737:chemistry; D012015:Reference Standards
chemical_list: C000591158:APL-1 peptide; D018834:Chaperonin 60; D010446:Peptide Fragments