Dynamic Monitoring of Newly Synthesized Proteomes: Up-Regulation of Myristoylated Protein Kinase A During Butyric Acid Induced Apoptosis

Abstract

Post-translational modification (PTM) is a highly dynamic yet precisely controlled process by which most eukaryotic proteins are chemically diversified. Many critical cellular responses are mediated through PTMs, which lead to modulation of enzyme activity, protein conformation, protein–protein interaction, and cellular localization. Analysis of these modifications at the proteome level could provide invaluable biological insight but remains a technically challenging undertaking. Traditionally, PTMs have been studied by standard molecular biology techniques involving tedious isolation of individual proteins and subsequent direct detection and analysis of amino acids bearing the modification. Recent advances in mass spectrometry, when combined with stable-isotope or metabolic labeling approaches, have enabled several large-scale studies of PTMs and their dynamics. These methods, however, analyze PTM changes of all proteins (old and new) present in the cell at the time of sampling and thus are only able to evaluate PTM dynamics at the ensemble level. With unnatural metabolic building blocks and in vivo compatible conjugation chemistries becoming increasingly available (Scheme 1), we sought to develop a proteomic strategy for the detection and identification of newly synthesized proteomes and their PTMs (Figure 1). We envisioned several advantages of studying the PTM dynamics of newly synthesized proteomes: 1) this method decreases the complexity of the proteome and enables the identification of PTM changes that occur in a predefined protein synthesis window; 2) it gives an accurate estimate of the time scale of different PTM events in transforming newly synthesized, modification-free proteins into mature functional entities; 3) it permits PTM analysis of primary protein synthesis responses to internal and external cues. To isolate a newly synthesized proteome, wemade use of BONCAT (bio-orthogonal noncanonical amino acid tagging), which uses the known methionine surrogates azidohomoalanine (AHA) and homopropargylglycine (HPG) for metabolic incorporation into newly synthesized proteins (Scheme 1, blue) and the corresponding alkyneor azide-modified biotin reporter (Scheme 1, bottom) for subsequent proteome isolation. To monitor dynamic changes of an PTM event, we fed growing cells with an azideor alkynecontaining sugar, fatty acid, or lipid building block (Scheme 1, orange). It should be noted that while our work was in progress, Hang and co-workers reported a tandem labeling and detection method to monitor the dynamic acylation of LCK (a tyrosine kinase) and its turnover. Their work focused on the study of single PTM events (i.e. protein palmitoylation) of a specific protein (i.e., LCK) in a proteome. The work herein, while conceptually similar, greatly expands the scope of this double metabolic incorporation strategy by successfully demonstrating, for the first Scheme 1. Methionine surrogates (blue) and unnatural metabolite PTM probes (orange) form bio-orthogonal pairs for compatible double metabolic incorporation. Those forming pairs are boxed in the same group. Azideand alkyne-containing fluorophore and biotin reporters are also shown (bottom).