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

Published

May 20, 2013

Author(s)

Junjun J. Chen, Richard Huang, Illarion Turko

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, 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 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 15N-labeled 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 SDS and following desalting/delipidation of the sample by chloroform/methanol precipitation with extra water-washing of 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.

Citation

Analytical Chemistry

Volume

85

Issue

12

Pub Type

Journals

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DOI Link

Keywords

mass spectrometry, quantification, partitioning, UCH-L1, frontal cortex, Alzheimer's disease

Citation

Chen, J. , Huang, R. and Turko, I. (2013), Mass Spectrometry Assessment of Ubiquitin Carboxyl-Terminal Hydrolase L1 Partitioning between Soluble and Particulate Brain Homogenate Fractions, Analytical Chemistry, [online], https://doi.org/10.1021/ac400831z (Accessed December 15, 2024)