A Novel Data Analysis Pipeline for Bottoms‐Up Mass Spectrometry Proteomic Analysis of Primary Tissues

Abstract

Modern mass spectrometry‐based proteomic studies require reliable, and robust data analysis for a comprehensive and quantitative profile of histone post‐translational modifications (PTM). Studies involving primary tissue samples face additional challenges in comparative sample analysis, due to various protein concentrations, multiple modification sites and modification states. A novel data analysis pipeline was developed in order to streamline the analysis process and normalize samples of varying concentration. This is relevant in the proteomic analysis of addiction, where various modification isoforms can correlate with different addiction phenotypes. A self‐administration model addiction study of methamphetamine (METH) addiction was used to develop this pipeline.Following nuclear protein isolation, proteins underwent tryptic digestion and histone H3 samples were derivatized with propionic anhydride. Identification, validation, and label‐free quantification of histone modifications was performed. Protein loading of different samples was normalized, using high abundance, proteotypic unmodified peptides. To localize changes in specific site and state, peptide isoforms for each site‐ and state‐specific modification were binned. Using this approach, we were able to identify histone modifications that underwent statistically significant changes in METH self‐administrated rat brain samples. In addition, we were able to identify histone modifications that are indicative of METH controllability in tested individual subjects. This data analysis pipeline has led to robust analysis of histone PTMs. Information on changes in site‐ and state‐specific histone modifications can be integrated with RNA‐seq data to elucidate driving chromatin pathways in the formation and maintenance of pathophysiological epigenetic states.Support or Funding InformationThis project was supported in part by the University of New Hampshire Hamel Center for Undergraduate Research.