TY - JOUR T1 - Optimization of Protocols for Detection of De Novo Protein Synthesis in Whole Blood Samples via Azide-Alkyne Cycloaddition. JF - J Proteome Res Y1 - 2020 A1 - Bowling, Heather L A1 - Kasper, Amanda A1 - Patole, Chhaya A1 - Venkatasubramani, Janani Priya A1 - Leventer, Sarah Parker A1 - Carmody, Erin A1 - Sharp, Kevin A1 - Berry-Kravis, Elizabeth A1 - Kirshenbaum, Kent A1 - Klann, Eric A1 - Bhattacharya, Aditi AB -

Aberrant protein synthesis and protein expression are a hallmark of many conditions ranging from cancer to Alzheimer's. Blood-based biomarkers indicative of changes in proteomes have long been held to be potentially useful with respect to disease prognosis and treatment. However, most biomarker efforts have focused on unlabeled plasma proteomics that include nonmyeloid origin proteins with no attempt to dynamically tag acute changes in proteomes. Herein we report a method for evaluating de novo protein synthesis in whole blood liquid biopsies. Using a modification of the "bioorthogonal noncanonical amino acid tagging" (BONCAT) protocol, rodent whole blood samples were incubated with l-azidohomoalanine (AHA) to allow incorporation of this selectively reactive non-natural amino acid within nascent polypeptides. Notably, failure to incubate the blood samples with EDTA prior to implementation of azide-alkyne "click" reactions resulted in the inability to detect probe incorporation. This live-labeling assay was sensitive to inhibition with anisomycin and nascent, tagged polypeptides were localized to a variety of blood cells using FUNCAT. Using labeled rodent blood, these tagged peptides could be consistently identified through standard LC/MS-MS detection of known blood proteins across a variety of experimental conditions. Furthermore, this assay could be expanded to measure de novo protein synthesis in human blood samples. Overall, we present a rapid and convenient de novo protein synthesis assay that can be used with whole blood biopsies that can quantify translational change as well as identify differentially expressed proteins that may be useful for clinical applications.

VL - 19 IS - 9 ER -