Mass Spectrometry Assessment of Ubiquitin Carboxyl-Terminal Hydrolase L1 Partitioning between Soluble and Particulate Brain Homogenate Fractions

Abstract

Partitioning of specific proteins between soluble and insoluble forms because of aggregation, membrane attachment, and (or) association with senile plaques and neurofibrillary tangles is a major feature of several neurodegenerative disorders, including Alzheimer's disease (AD). Ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) is an example of a neuron-specific protein which displays two different dimerization-dependent catalytic activities and can be farnesylated for membrane attachment, oxidized, and truncated. Decreased levels of soluble UCH-L1 are inversely proportional to the number of neurofibrillary tangles. Further assessment of a link between UCH-L1 function and the pathogenesis of AD requires an analytical method to separately quantify different UCH-L1 forms. In the present study, we have developed a multiple reaction monitoring (MRM) assay to measure UCH-L1 in the high-speed supernatant and pellet of frontal cortex homogenate. The well-characterized (15)N-labeled quantification concatamer (QconCAT) carrying prototypic tryptic peptides of UCH-L1 was used as an internal standard. The composed protocol of frontal cortex processing includes solubilization and reduction/alkylation of proteins in the presence of 1% sodium dodecyl sulfate (SDS) and following with desalting/delipidation of the sample by chloroform/methanol precipitation with extra water washing of the protein pellet. The measurements were performed for frontal cortex samples from control and severe AD donors. The proposed workflow can be recommended for quantification of partitioning of other proteins of interest.