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Mass spectrometry quantification of PICALM and AP180 in human frontal cortex and neural retina
Junjun Chen1, Irina A Pikuleva, Illarion V Turko
1Institute for Bioscience and Biotechnology Research, University of Maryland, Rockville, MD 20850, USA; Biomolecular Measurement Division, National Institute of Standards and Technology, Gaithersburg, MD 20899, USA.
Abstract:
Recent genome-wide association studies have suggested that endocytic factors, such as phosphatidylinositol-binding clathrin assembly protein (PICALM), may be implicated in the development of Alzheimer disease (AD). The cellular functions of PICALM are in line with this possibility: (i) PICALM is involved in regulation of amyloid-β levels and (ii) PICALM is important for a presynaptic function, which is diminished in AD. To facilitate the analysis of PICALM, we developed a quantitative method to assess the expression level of PICALM in various biological samples. For this purpose, a stable isotope-labeled quantification concatamer (QconCAT) of PICALM was designed, expressed, purified, and characterized. The PICALM QconCAT was first used as an internal standard in a multiple reaction monitoring assay to measure PICALM concentrations in the human frontal cortex, a tissue strongly affected by AD. A second endocytic factor that is highly homologous to PICALM and also functions in clathrin-mediated endocytosis, clathrin coat assembly protein AP180, was quantified as well. Because age-related macular degeneration shares several clinical and pathological features with AD, the measurements were then extended to human normal neural retina. Overall, the developed method is suitable for PICALM and AP180 quantitative analysis in various biological samples of interest.
Insights
Researchers developed a new method to quantify phosphatidylinositol-binding clathrin assembly protein (PICALM) and AP180. This tool aids Alzheimer disease (AD) research by measuring these endocytic factors in brain and retinal tissues.
Area of Science:
- Neuroscience
- Molecular Biology
- Biochemistry
Background:
- Genome-wide association studies suggest endocytic factors, like PICALM, are linked to Alzheimer disease (AD).
- PICALM's roles in amyloid-β regulation and presynaptic function align with its potential involvement in AD pathogenesis.
- Understanding PICALM expression is crucial for investigating its role in neurological disorders.
Purpose of the Study:
- To develop and validate a quantitative method for measuring PICALM expression levels in biological samples.
- To apply this method for quantifying PICALM and AP180 in human brain and retinal tissues.
- To facilitate further research into the role of PICALM in AD and related conditions.
Main Methods:
- Designed, expressed, and purified a stable isotope-labeled quantification concatamer (QconCAT) of PICALM.
- Utilized the PICALM QconCAT as an internal standard in a multiple reaction monitoring (MRM) assay.
- Quantified PICALM and the homologous endocytic protein AP180 in human frontal cortex and neural retina samples.
Main Results:
- Successfully developed and characterized a PICALM QconCAT for quantitative proteomic analysis.
- Established a robust MRM assay for accurate measurement of PICALM and AP180 concentrations.
- Demonstrated the method's applicability to AD-affected brain tissue and neural retina.
Conclusions:
- The developed QconCAT-based MRM method provides a reliable tool for quantifying PICALM and AP180.
- This quantitative approach is suitable for analyzing these endocytic factors in various relevant biological samples.
- The method supports further investigation of PICALM's role in Alzheimer disease and age-related macular degeneration.
