Mature proteins of LAG-3 with or without mutations were digested with chymotrypsin followed by data-dependent LC-MS/MS analysis

ABSTRACT: The FLAG-tagged soluble monomeric proteins of LAG-3-WT, LAG-3-V20A, and LAG-3-L14Q/V20A were immunoprecipitated from culture supernatants of Plat-E cells transfected with the corresponding plasmid vectors using ANTI-FLAG M2 Magnetic Beads. Proteins on the beads were digested with chymotrypsin (Roche) followed by reduction and alkylation. The peptides were subjected to data-dependent LC-MS/MS analysis using an EASY-nLC 1200 UHPLC connected to a Q Exactive Plus mass spectrometer through a nanoelectrospray ion source (Thermo Fisher Scientific). Raw data were directly analyzed against the SwissProt database restricted to H. sapiens supplemented with predicted amino acid sequences of LAG-3 with or without mutations starting from A18 to L29 using Proteome Discoverer version 2.3 (Thermo Fisher Scientific) with Sequest HT search engine. The search parameters were as follows: (a) chymotrypsin as an enzyme with up to two missed cleavages; (b) precursor mass tolerance of 10 ppm; (c) fragment mass tolerance of 0.02 Da; (d) carbamidomethylation of cysteine as a fixed modification; and (e) acetylation of protein N-terminus and oxidation of methionine as variable modifications. Peptides were filtered at a false discovery rate of 1% using the percolator node.

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Hidetaka Kosako 

PROVIDER: PXD023929 | JPOST Repository | Mon Apr 11 00:00:00 BST 2022

REPOSITORIES: jPOST