Developing a Lysosomal Panel for Biomarker Discovery in Neurodegenerative Diseases Using PRM

摘要

Here we present the development of a PRM method by identifying a target panel of proteins representing an intracellular biological system and setting up a scheduled targeted MS/MS method for quantitation of these proteins. Protein identification was performed by using nano-HPLC MS/MS. Two methods for selecting precursor ions were compared; a combination of least and most intense ions inclusion methods and a sandwich method. The two methods resulted in the identification of a similar set of proteins. However, the number of peptides identified was exceedingly higher using a combination of the least and most intense ions methods. The drawback of this approach is the necessary two injections and separations of the sample. From the least and most intense ions methods we identified a set of 16 relevant proteins and selected 38 proteotypic tryptic peptides. Isotope labeled peptides corresponding to the selected tryptic peptides was acquired and used to develop a micro-HPLC scheduled targeted MS/MS method. Suitable fragment ions were identified and used for monitoring the repeatability from injection to injection for tryptic and isotope labeled peptides. Also four different methods for digestion of cerebrospinal fluid were compared. The tryptic fragment ion peak areas generated were overall less using 50 and 100μL cerebrospinal fluid and the SMART digest kit as compared to using 100μL cerebrospinal fluid and trypsin or trypsin/LysC for digestion. Fragment ion peak areas of the isotope labeled peptides were slightly higher for the SMART 50 group. In part this might explain the low ratio and higher variability for digesting 50μL cerebrospinal fluid with the SMART digest kit. Reduction and alkylation was not part of procedure for preparing the SMART digest samples, therefore peptides including cysteines were excluded from SMART digest samples in the analysis. Overall the lowest variation for the peptides included in the method, in regard to the ratio of tryptic to isotope labeled peptide, was achieved using trypsin and trypsin/LysC. However, the variation was peptide dependent. Any of the methods employed for digestion could likely be further optimized to improve the repeatability of single or several peptides. The difficulty exists in having one optimal digestion method for digesting such a diverse array of proteins. We are currently moving forward to utilize the developed method to explore the level of the Endo-Lysosomal panel of proteins in several case-control cohorts.