Targeted Quantitative Proteomic Approach for High-Throughput Quantitative Profiling of Small GTPases in Brain Tissues of Alzheimer’s Disease Patients

Neurodegenerative
disorders, including Alzheimer’s disease
(AD), are prevalent among the elderly. Small GTPases of the Ras superfamily
are essential regulators of intracellular trafficking and signal transduction.
In this study, we develop a targeted quantification method for small
GTPase proteins, where the method involves scheduled multiple-reaction
monitoring analysis and the use of synthetic stable isotope-labeled
peptides as internal standards or surrogate standards. We further
applied this method to examine the altered expression of small GTPase
proteins in post-mortem frontal cortex tissues from AD patients with
different degrees of disease severity. We were able to achieve sensitive
and reproducible quantifications of 80 small GTPases in brain tissue
samples from 15 patients. Our results revealed substantial up-regulations
of several synaptic GTPases, i.e., RAB3A/C, RAB4A/B, and RAB27B, in
tissues from patients with higher degrees of AD pathology, suggesting
that aberrant synaptic trafficking may modulate the progression of
AD. The method should be generally applicable for high-throughput
targeted quantification of small GTPase proteins in other tissue and
cellular samples.