A novel multi-faceted method for large-scale characterization and relative quantitation of citrullinated proteins for biological samples and its application to Alzheimer's disease

摘要

ABSTRACT Protein citrullination as an enzymatic post-translational modification (PTM) plays important roles in the onset and progression of many diseases, including rheumatoid arthritis, neurodegenerative diseases, diabetes and cardiovascular problems. Current citrullination studies have been primarily relying on immunohistochemistry and Western blotting techniques, and suffer from lack of effective methods for qualitative and quantitative analysis of citrullinated proteins in clinically relevant samples, limiting our understanding of this disease-related PTM. Mass spectrometry (MS) has become a powerful tool for proteomics and site-specific analysis of many PTMs including acetylation, phosphorylation and glycosylation. However, MS-based citrullination analysis presents significant challenge due to the lack of effective methods for specific enrichment of citrullinated proteins in biological samples, and tandem-MS based approaches enabling accurate identification of citrullinated peptides and reliable assignment of citrullination site. The primary goal of this proposal is to develop a multi-faceted approach integrating biotin tag-assisted enrichment method, isobaric tag-based quantitative strategy with MS- based technologies for simultaneous enrichment, large-scale characterization and relative quantitation of citrullinated proteins in biological samples, and apply this multi-faceted approach to investigate the roles of citrullination in Alzheimer’s disease (AD). We propose the following specific aims: Specific Aim 1 – To develop a novel biotin tag-assisted MS-based method for large-scale characterization of citrullinated proteins in biological samples. Specific Aim 2 – To establish a multi-faceted method integrating multiplexed dimethylated leucine (DiLeu)-based quantitation strategy with biotin-assisted MS-based method for simultaneous characterization and relative quantitation of citrullinated proteins from biological samples. Specific Aim 3 – To apply the newly-developed multi-faceted method for citrullinome analysis of cerebrospinal fluid (CSF) samples from age-matched asymptomatic cognitively-healthy adults, preclinical stage 3 individuals and patients with mild cognitive impairment (MCI) and AD. Collectively, our proposed experiments will develop a novel MS-based method enabling large-scale and relative quantitation of citrullinated proteins in complicated biological samples. This is also the first time that the roles of citrullination in AD will be studied, advancing our knowledge of AD and providing an opportunity for new AD biomarker discovery.