Transforming chemical proteomics into high-throughput method using SP2E workflow.

Protein post-translational modifications (PTMs) play a critical role in regulation of protein catalytic activity, localization and protein-protein interactions. An attachment of PTMs onto proteins significantly diversify their structure and function resulting in so called proteoforms. However, the sole identification of post-translationally modified proteins, which are often cell type and disease specific, is still a highly challenging task. Sub-stoichiometric amounts and modification of low abundant proteins necessitate purification or enrichment of the modified proteins. Introduction of the mass spectrometry-based chemical proteomic strategy has enabled to screen protein PTMs with increased throughput, but the sample preparation has remained highly time consuming and tedious. Here, we report an optimized workflow for enrichment of PTM proteins in 96-well plate format which could be extended to robotic automation. This platform allows to significantly lower the input of total protein, which opens up the possibility to screen specialized and difficult to culture cell lines in high-throughput manner. The presented SP2E protocol is robust, time- and cost-effective as well as suitable for large-scale screening of proteoforms. Application of the SP2E protocol will thus enable the characterization of proteoforms in various processes such as neurodevelopment, neurodegeneration and cancer. This may contribute to an overall acceleration of the recently launched Human Proteoform Project.