Evaluating Combined Precursor Isotopic Labeling and Isobaric Tagging Performance on Orbitraps To Study the Peripheral Proteome of Alzheimer’s Disease

Combined
precursor isotopic labeling and isobaric tagging (cPILOT)
is an enhanced multiplexing strategy currently capable of analyzing
up to 24 samples simultaneously. This capability is especially helpful
when studying multiple tissues and biological replicates in models
of disease, such as Alzheimer’s disease (AD). Here, cPILOT
was used to study proteomes from heart, liver, and brain tissues in
a late-stage amyloid precursor protein/presenilin-1 (APP/PS-1) human
transgenic double-knock-in mouse model of AD. The original global
cPILOT assay developed on an Orbitrap Velos instrument was transitioned
to an Orbitrap Fusion Lumos instrument. The advantages of faster scan
rates, lower limits of detection, and synchronous precursor selection
on the Fusion Lumos afford greater numbers of isobarically tagged
peptides to be quantified in comparison to the Orbitrap Velos. Parameters
such as LC gradient, m/z isolation
window, dynamic exclusion, targeted mass analyses, and synchronous
precursor scan were optimized leading to >600 000 PSMs,
corresponding
to 6074 proteins. Overall, these studies inform of system-wide changes
in brain, heart, and liver proteins from a mouse model of AD.