Post‐Translational Modification Profiling—a High‐Content Assay for Identifying Protein Modifications in Mammalian Cellular Systems

Abstract

Protein microarrays are extremely useful for detecting substrates of protein kinases, substrates of ubiquitin ligases and other post translational modifications. The ability to screen binding interactions as well as post-translational modifications of thousands of proteins at once has improved our ability to identify their targets. Utilizing such systems in combination with functional mammalian cell extracts that preserve enzymatic activity offers advantages in identifying semi-quantitative changes of these interactions in the context of specific cellular conditions. This article provides a detailed procedure for setting up an extract-based activity assay for high content detection of protein post-translation modifications. It also provides basic guidelines for data analysis.

Post-translational modification profiling – A novel tool for mapping the protein modification landscape in cancer

“…In the next sections, we shall discuss in detail the recently developed PTM profiling approach [30][31][32][33] in the context of specific biological questions related to analysis of the enzymatic machinery and substrates of the ubiquitin system. We shall further illustrate its potential for analyzing the ubiquitin system in the context of cancer research.…”

Protein microarray applications: Autoantibody detection and posttranslational modification

“…In recent attempts to enhance the comprehension of PTMs, protein microarrays have been used as a platform to understand enzymatic reactions in vitro. It has also been used to perform direct studies of protein modifications using cultured cellular extracts surpassing the need to use labour intensive protein purification steps . For instance, Spruck and co‐workers fabricated protein array with around 8000 human recombinant proteins, which can be used to perform “on chip” reaction using either purified enzyme, cell lysates, or biological fluids .…”

Computational Modeling of Lysine Post-Translational Modification: An Overview

“…For example, it has been observed that nearly 60% class PTMs of protein succinylation sites are surrounded or overlapped with protein acetylation sites [8]. These PTMs can also affect the physicochemical properties of proteins, which can provide a mechanism for the dynamic regulation of molecular, self-assembly [9]. The PTMs have been found in the all types of proteins such as the structural proteins, plasma membrane receptors and nuclear transcription factors (Figure 1).…”