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

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.