Optimization of Protocols for Detection of de Novo Protein Synthesis in Whole Blood Samples via Azide-Alkyne Cycloaddition

Heather L. Bowling, Amanda Kasper, Chhaya Patole, Janani Priya Venkatasubramani, Sarah Parker Leventer, Erin Carmody, Kevin Sharp, Elizabeth Berry-Kravis, Kent Kirshenbaum, Eric Klann, Aditi Bhattacharya

Research output: Contribution to journalArticle

Abstract

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.

Original languageEnglish (US)
Pages (from-to)3856-3866
Number of pages11
JournalJournal of Proteome Research
Volume19
Issue number9
DOIs
StatePublished - Sep 4 2020

Keywords

  • blood-based biomarkers
  • copper(I)-catalyzed click chemistry
  • de novo protein synthesis
  • ethylenediaminetetraacetic acid
  • labeled proteomics
  • whole blood

ASJC Scopus subject areas

  • Biochemistry
  • Chemistry(all)

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  • Cite this

    Bowling, H. L., Kasper, A., Patole, C., Venkatasubramani, J. P., Leventer, S. P., Carmody, E., Sharp, K., Berry-Kravis, E., Kirshenbaum, K., Klann, E., & Bhattacharya, A. (2020). Optimization of Protocols for Detection of de Novo Protein Synthesis in Whole Blood Samples via Azide-Alkyne Cycloaddition. Journal of Proteome Research, 19(9), 3856-3866. https://doi.org/10.1021/acs.jproteome.0c00299