Dataset Information

FASIL-MS: An Integrated Proteomic and Bioinformatic Workflow to Universally Quantitate In vivo-acetylated Positional Isomers

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

Vitko Dijana D Májek Peter P Schirghuber Erika E Kubicek Stefan S Bennett Keiryn L KL

Journal of proteome research 20160705 8

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 considera ...[more]

PMID: 27302567

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Project description:BACKGROUND & AIMS- More frequent interaction of bacteria with the colonic epithelium is associated with ulcerative colitis (UC). The identities of all proteins which promote bacterial clearance in colonic epithelial cells are unknown. Previously, we discovered that dCAP-D3 (Chromosome Associated Protein-D3), regulates responses to bacterial infection. We examined whether CAP-D3 promotes bacterial clearance in human colonic epithelium. METHODS- Clearance of Salmonella or adherent-invasive Escherichia coli LF82 was assessed by gentamycin protection assays in HT-29 and Caco-2 cells expressing CAP-D3 shRNA. CAP-D3 levels in colonic epithelial cells from healthy and UC patient tissues were analyzed by immunoblot. RNA-sequencing identified bacterially-induced CAP-D3 target genes. The role of CAP-D3 target genes in bacterial clearance was analyzed by gentamycin protection assays, immunofluorescent staining, and by using pharmacologic inhibitors. RESULTS- CAP-D3 expression was reduced in colonic epithelial cells from UC patients with active disease. Reduction of CAP-D3 expression inhibited autophagy and decreased intracellular bacterial clearance. The components of the heterodimeric SLC7A5/SLC3A2 amino acid transporter were identified as CAP-D3 target genes; their levels increased in infected, CAP-D3 deficient cell lines and in cells from UC patients. In HT-29 cells, this resulted in earlier SLC7A5 recruitment to Salmonella-containing vacuoles, increased mTOR activity, and enhanced bacterial survival. Inhibition of SLC7A5/SLC3A2 or mTOR activity rescued the bacterial clearance defect in CAP-D3 deficient cells. CONCLUSIONS- CAP-D3 attenuates amino acid transporter transcription to promote bacterial autophagy in colon epithelial cells. CAP-D3 protein levels are decreased in patients with active UC, suggesting that CAP-D3 is a potential therapeutic target to restore mucosal homeostasis in UC patients. Three RNA samples from 3 independent experiments including timepoints taken at 0, 0.5 and 7 hours post-infection were analyzed on a bioanalyzer for quality; one of the 0.5 hour post-infection samples was excluded at this time due to poor RNA purity. Directional, cDNA libraries made from cellular mRNAs were generated from the other 8 samples and sequenced (paired-end sequencing of 100 bp reads) in the Genomics Core at the University of Chicago on an Illumina HiSeq2000.

			Action	DRS
	BSA-D3-NAS-trypsin-1.msf	Msf
	BSA-D3-NAS-trypsin-1.raw	Raw
	BSA-D3-NAS-trypsin-2.msf	Msf
	BSA-D3-NAS-trypsin-2.raw	Raw
	BSA-D3-NAS-trypsin-3.msf	Msf