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Histone post‐translational modifications (PTMs) play various roles in chromatin‐related cellular processes, and comprehensive analysis of these combinatorial PTMs at the intact protein level by top‐down proteomics is the method of choice to reveal their crosstalk and biological functions. Here, we report our top‐down characterization of the core histones from mouse fibroblasts cells NIH/3T3, which is a classic model used in many kinds of research. With nanoRPLC‐MS/MS analysis and ProteinGoggle database search, 547 protein species were identified with spectrum‐level FDR ≤ 1%, where PTMs in 51 protein species were unambiguously localized with PTM scores ≥1. High‐resolution MS/MS data also allowed the unambiguous identification of acetylation instead of trimethylation. This study presents a general picture of combinatorial PTMs of mouse core histones, which serves as a basic reference for all future related biological studies. [ABSTRACT FROM AUTHOR]

Neutral loss and internal product ions have been found to be significant in both peptide and protein tandem mass spectra and they have been proposed to be included in database search and for protein identification. In addition to common canonical b/y ions in collision-based dissociation or c/z ions in electron-based dissociation, inclusion of neutral loss and internal product ions would certainly make better use of tandem mass spectra data; however, their ultimate utility for protein identification with false discovery rate control remains unclear. Here we report our proteome-level utility benchmarking of neutral loss and internal product ions with tandem mass spectra of intact E. coli proteome. Utility of internal product ions was further evaluated at the protein level using selected tandem mass spectra of individual E. coli proteins. We found that both neutral loss and internal products ions do not have direct utility for protein identification when they were used for scoring of P Score; but they do have indirect utility for provision of more canonical b/y ions when they are included in the database search and overlapping ions between different ion types are resolved. Biological significance Tandem mass spectrometry has evolved to be a state-of-the-art method for characterization of protein primary structures (including amino acid sequence, post-translational modifications (PTMs) as well as their site location), where full study and utilization tandem mass spectra and product ions are indispensable. This primary structure information is essential for higher order structure and eventual function study of proteins. [ABSTRACT FROM AUTHOR]