In vitro and in cellulae methods for determining the target protein SUMOylation.

Post-translational modifications (PTMs) provide a critical means of calibrating the functional proteome and, thus, are extensively utilized by the eukaryotes to exert spatio-temporal regulation on the cellular machinery rapidly. Ubiquitination and phosphorylation are examples of the well-documented PTMs. SUMOylation, the reversible conjugation of the Small Ubiquitin-related MOdifier (SUMO) at a specific lysine residue on a target protein, bears striking similarity with ubiquitination and follows an enzymatic cascade for the attachment of SUMO to the target protein. Unlike Ubiquitination, SUMOylation can modulate the target protein's structure, stability, activity, localization, and interaction. Thus, SUMOylation regulates cellular events such as signal transduction, cell-cycle progression, transcription, nucleocytoplasmic transport, and stress responses. Accordingly, deregulation of SUMOylation is an avenue for diseases, which makes the investigation of SUMO and its substrates within the cell essential. However, the low extent of SUMOylation has posed a significant challenge in detecting SUMO modification within the cell. Bioinformatics tools can help predict SUMOylation, and mass-spectrometric analysis can identify a pool of cellular protein SUMOylome. Nevertheless, the biochemical methods for observing the enhanced level of in vitro SUMOylation help validate protein SUMOylation, critical lysine(s) utilized in the process, and its effect on substrate protein function. This chapter provides a detailed account of biochemical methods commonly utilized to detect SUMOylated proteins that are central for understanding the biological functions and mechanism of regulation of SUMO targets.

Traceless cysteine-linchpin enables precision engineering of lysine in native proteins.

The maintenance of machinery requires its operational understanding and a toolbox for repair. The methods for the precision engineering of native proteins meet a similar requirement in biosystems. Its success hinges on the principles regulating chemical reactions with a protein. Here, we report a technology that delivers high-level control over reactivity, chemoselectivity, site-selectivity, modularity, dual-probe installation, and protein-selectivity. It utilizes cysteine-based chemoselective Linchpin-Directed site-selective Modification of lysine residue in a protein (LDMC-K). The efficiency of the end-user-friendly protocol is evident in quantitative conversions within an hour. A chemically orthogonal C-S bond-formation and bond-dissociation are essential among multiple regulatory attributes. The method offers protein selectivity by targeting a single lysine residue of a single protein in a complex biomolecular mixture. The protocol renders analytically pure single-site probe-engineered protein bioconjugate. Also, it provides access to homogeneous antibody conjugates (AFC and ADC). The LDMC-K-ADC exhibits highly selective anti-proliferative activity towards breast cancer cells.