Absolute Quantitation of Low
Abundance Plasma APL1β
peptides at Sub-fmol/mL Level by SRM/MRM without Immunoaffinity Enrichment
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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 C<sub>18</sub>-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