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

Abstract

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 diversifies 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. Although the introduction of the mass spectrometry-based chemical proteomic strategy has enabled to screen protein PTMs with increased throughput, 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 can be possible extended to robotic automatization. This platform allows to significantly lower the input of total protein, which opens up the opportunity 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 and may contribute to an overall acceleration of the recently launched Human Proteoform Project.

XGboost-Ampy: Identification of AMPylation Protein Function Prediction Using Machine Learning

“…It controls many biological processes, from sickness in different animal species to regulation of bacterial nitrogen metabolism to signaling pathway regulation [13] - [14] . The regulation of neurodevelopment and neurodegeneration by AMPylation has also been established [15] . Methods used to identify PTM sites need a lot of time, money, and effort to implement.…”