FASIL-MS: An Integrated Proteomic and Bioinformatic Workflow to Universally Quantitate In vivo-acetylated Positional Isomers
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ABSTRACT: 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 alter cell proliferation that can lead to disease progression are of considerable medical interest. In this study, stable isotope labelling with N-acetoxy-D3-succinimide (D3-NAS) was utilised to efficiently derivatise 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 derivatisation to filter-aided sample preparation (FASP). Additionally, the mass spectrometry method was adapted to simultaneously co-isolate and concurrently fragment all differentially-H3/D3-acetylated histone peptide clusters. The combination of these multi-plexed MS2 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 labelling coupled to mass spectrometry. FASIL-MS enables the universal and site-specific quantitation of peptides with multiple in vivo-acetylated lysine residues.
INSTRUMENT(S): LTQ Orbitrap Velos
ORGANISM(S): Homo Sapiens (human) Bos Taurus (bovine)
TISSUE(S): Cell Culture, Epithelial Cell Of Cervix
DISEASE(S): Cervix Carcinoma
SUBMITTER: Dijana Vitko
LAB HEAD: Keiryn L. Bennett
PROVIDER: PXD003611 | Pride | 2016-06-16
REPOSITORIES: Pride
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