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Peptide affinity chromatography identifies proteins that bind to N‐terminus of NFAT5
Author(s) -
Izumi Yuichiro,
Jinxi Li,
Burg Mauice B.,
Ferraris Joan D.
Publication year - 2013
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.27.1_supplement.728.3
Subject(s) - peptide , stable isotope labeling by amino acids in cell culture , proteome , chemistry , affinity chromatography , biochemistry , cytoplasm , orbitrap , amino acid , microbiology and biotechnology , biology , proteomics , chromatography , mass spectrometry , gene , enzyme
Nuclear Factor of Activated T‐Cells 5 (NFAT5) induces transcription of osmoprotective genes in response to hypertonicity. NFAT5 is associated with many other proteins, only a few of which have been identified. In order to identify additional proteins that interact with NFAT5, we performed peptide affinity chromatography. Biotin‐tagged peptides were synthesized, each containing 20 amino acids from a region in the N‐terminus of NFAT5 predicted to be intrinsically disordered. HEK293 cells were grown in light, medium, or heavy SILAC medium, then incubated at 200, 300, or 500 mOsm (NaCl varied), respectively, for 30 min. Cytoplasmic or nuclear extracts from the different osmolalities were combined and incubated with peptides and streptavidin‐coated magnetic beads to isolate proteins associated with the peptides. Proteins eluted from the beads were analyzed on an Eksigent nanoLC Ultra coupled with a Thermo LTQ‐Orbitrap Velos mass spectrometer. LC‐MS/MS files were searched via Proteome Discoverer (PD) against Sprot human database for peptide and protein identification. Numerous proteins were reproducibly identified in biological replicates associated with peptide baits 131 – 150, 161 – 180, 191 – 210 and 206 – 225 in nuclear and/or cytoplasmic fractions. Peptide affinity chromatography revealed proteins that are associated with NFAT5 and that presumably regulate it in response to hypertonicity.