Protocol for proteomic profiling of hypusinated protein candidates using a clickable spermidine probe

Hypusination is a unique posttranslational modification in which deoxyhypusine synthase (DHPS) transfers an aminobutyl moiety from spermidine to specific lysine residues, followed by deoxyhypusine hydroxylase (DOHH)-mediated hydroxylation. Here, we present a protocol that enables proteome-wide identification of candidate hypusinated proteins. We describe steps for the synthesis of a clickable alkynyl-spermidine probe, DHPS-dependent metabolic labeling in cells, click chemistry-mediated biotinylation, streptavidin-based enrichment, and subsequent mass spectrometry analysis of probe-labeled proteins. We also describe the procedures for data processing and statistical analysis. For complete details on the use and execution of this protocol, please refer to Zhang et al.¹.