FASIL-MS: An Integrated Proteomic and Bioinformatic Workflow To Universally Quantitate In Vivo-Acetylated Positional Isomers.

Dynamic changes in histone post-translational modifications (PTMs) regulate gene transcription leading to fine-tuning of biological processes such as DNA replication and cell cycle progression. Moreover, specific histone modifications constitute docking sites for recruitment of DNA damage repair proteins and mediation of subsequent cell survival. Therefore, understanding and monitoring changes in histone PTMs that can alter cell proliferation and thus lead to disease progression are of considerable medical interest. In this study, stable isotope labeling with N-acetoxy-D3-succinimide (D3-NAS) was utilized to efficiently derivatize unmodified lysine residues at the protein level. The sample preparation method was streamlined to facilitate buffer exchange between the multiple steps of the protocol by coupling chemical derivatization to filter-aided sample preparation (FASP). Additionally, the mass spectrometry method was adapted to simultaneously coisolate and subsequently cofragment all differentially H3/D3-acetylated histone peptide clusters. Combination of these multiplexed MS(2) spectra with the implementation of a data analysis algorithm enabled the quantitation of each and every in vivo-acetylated DMSO- and SAHA-treated H4(4-17) and H3(18-26) peptide. We have termed our new approach FASIL-MS for filter-aided stable isotopic labeling coupled to mass spectrometry. FASIL-MS enables the universal and site-specific quantitation of peptides with multiple in vivo-acetylated lysine residues. Data are available via ProteomeXchange (PXD003611).