Molecular Basis of the mechanisms controlling MASTL - Proteomics analysis of MASTL substrate proteome and phosphorylation state

ABSTRACT: As part of our study on human MASTL (microtubule-associated serine/threonine kinase-like), we used mass spectrometry (MS)-based proteomics in the aim to unravel novel aspects about MASTL activation, regulation and potential substrates. Specifically, we performed a phosphoproteomics-based kinase assay (essentially as in Xue et al. 2014) to identify MASTL substrates and analyzed different MASTL constructs to generate a “phosphorylation map” of the MASTL protein. siKALIP (Stable Isotope Labelled Kinase Assay-Linked Phosphoproteomics) Three MASTL variants, which we termed MASTL (full-length), MASTL450 (short) and Bonsai (truncated), were expressed in HEK293 cells and isolated using a tandem Strep and His-tag affinity purifications. For the in vitro kinase assay we used a dephosphorylated cell extract from interphase HEK293 cells to perform a slightly modified version of the siKALIP (described in Xue et al. 2014). Samples included an untreated basal sample, a dephosphorylated sample and samples for each of the three MASTL constructs. For label-free quantification, reactions were performed in quadruplicates for each MASTL construct. Explanation for MS raw files with MASTL construct included: FL: MASTL full-length. 450short: Shorter (consisting of the first 450 amino acid residues) MASTL. Bonsai: Truncated MASTL (consisting of amino acid residues 35-112, 113-180 and 728-879). MASTL phosphorylation map To analyse the phosphorylation state of MASTL we used isolated protein from an interphase HEK293 cell culture, either alone or incubated with mitotic extracts from HEK293 cells. Samples of different MASTL constructs were used for in-gel digestion and analysed by MS. Explanation for MS raw files with MASTL construct included: FL: MASTL full-length (“INACTIVE” indicates kinase-dead, “Dephos” indicates dephosphorylated FL protein, “Mit” indicates incubated with mitotic extracts). 450: Shorter (450 amino acids) MASTL (“KD” indicates kinase-dead). BONSAI: Truncated MASTL (“KD” indicates kinase-dead).

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Anna-Kathrine Pedersen  

LAB HEAD: Guillermo Montoya

PROVIDER: PXD007991 | Pride | 2020-01-09

REPOSITORIES: Pride