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.
