MassIVE MSV000092218

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Interaction with IP6K1 supports pyrophosphorylation of substrate proteins by the inositol pyrophosphate 5-IP7

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Description

HEK293T cells transiently transfected to express either SFB-tagged GFP or SFB-tagged IP6K1 were lysed in NETN buffer (20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40) containing protease and phosphatase inhibitor cocktails (Merck), at 4 degree C for 60 min. Cell debris were removed by centrifugation at 14000 x g for 10 min, and cell lysates were incubated with streptavidin-sepharose beads (Cytiva Life Sciences) for 1 h at 4 degree C with end-over-end rotation. The bound protein complexes were washed thrice with NETN buffer and then eluted with 2 mg/mL biotin (Merck) for 90 min at 4 degree C. The eluates were incubated with S-protein agarose beads (Novagen) for 1 h at 4 degree C and then washed thrice with NETN buffer. Proteins bound to S-protein agarose beads were boiled in 2x SDS sample buffer for 5 min, loaded on a 12% SDS polyacrylamide gel, allowed to run into the resolving gel up to 1 cm, and visualized by Coomassie Brilliant Blue staining. All proteins in the sample were excised in one gel slice and sent for mass spectrometry analysis to Taplin Biological Mass Spectrometry Facility at Harvard University, USA. Briefly, gel pieces were subjected to in-gel digestion with sequencing-grade trypsin (Promega), and the extracted peptides were resolved by reverse phase HPLC. Eluted peptides were subjected to electrospray ionization (ESI) and then allowed to enter an LTQ Orbitrap Velos Pro ion-trap mass spectrometer (Thermo Fisher Scientific). Peptides were detected, isolated, and fragmented to produce a tandem mass spectrum of specific fragment ions for each peptide. Peptide sequences (and hence protein identity) were determined by matching protein databases from UniProt (https://www.uniprot.org/taxonomy/10090) with the acquired fragmentation pattern by the software program, Sequest (Thermo Fisher Scientific). [doi:10.25345/C5C24QZ0C] [dataset license: CC0 1.0 Universal (CC0 1.0)] [doi:10.25345/C5K64B36R] [dataset license: CC0 1.0 Universal (CC0 1.0)]

Keywords: IP6K1 ; 5-IP7

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Principal Investigators:
(in alphabetical order) 		Rashna Bhandari, Centre for DNA Fingerprinting and Diagnostics, India
Submitting User: akruti26
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