Molecular basis of Tousled-Like-Kinase 2 (TLK2) activation – LC-MS/MS-based analysis of HEK293-expressed TLK2 phosphorylation state

In this study on human Tousled-Like-Kinase 2 (TLK2) mass spectrometry-based analysis was applied as part of a molecular characterization of TLK2 in the aim to increase the understanding of the mode of activation of this protein. Specifically, different TLK2 constructs were expressed analyzed to generate a “phosphorylation map” of the TLK2 protein. To analyse the phosphorylation state of TLK2 we used recombinant protein purified from HEK293. Samples of different TLK2 constructs were used for in-gel digestion and analysed by mass spectrometry. All samples were prepared and run in triplicates for label-free quantification. Explanation for MS raw files with TLK2 construct included: KD: Kinase-dead TLK2 construct with mutation D613A, N-terminal His- and Strep-tag. wt: Active form of TLK2, canonical sequence, N-terminal His- and Strep-tag. full: Full-length canonical sequence (1-772), N-terminal His- and Strep-tag. trunc: Truncated TLK2 sequence (191-772), N-terminal His- and Strep-tag.