%0 Journal Article %J J Proteome Res %D 2020 %T Optimization of Protocols for Detection of De Novo Protein Synthesis in Whole Blood Samples via Azide-Alkyne Cycloaddition. %A Bowling, Heather L %A Kasper, Amanda %A Patole, Chhaya %A Venkatasubramani, Janani Priya %A Leventer, Sarah Parker %A Carmody, Erin %A Sharp, Kevin %A Berry-Kravis, Elizabeth %A Kirshenbaum, Kent %A Klann, Eric %A Bhattacharya, Aditi %X

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.

%B J Proteome Res %V 19 %P 3856-3866 %8 2020 Sep 04 %G eng %N 9 %R 10.1021/acs.jproteome.0c00299 %0 Journal Article %J Nat Commun %D 2019 %T Altered steady state and activity-dependent de novo protein expression in fragile X syndrome. %A Bowling, Heather %A Bhattacharya, Aditi %A Zhang, Guoan %A Alam, Danyal %A Lebowitz, Joseph Z %A Bohm-Levine, Nathaniel %A Lin, Derek %A Singha, Priyangvada %A Mamcarz, Maggie %A Puckett, Rosemary %A Zhou, Lili %A Aryal, Sameer %A Sharp, Kevin %A Kirshenbaum, Kent %A Berry-Kravis, Elizabeth %A Neubert, Thomas A %A Klann, Eric %X

Whether fragile X mental retardation protein (FMRP) target mRNAs and neuronal activity contributing to elevated basal neuronal protein synthesis in fragile X syndrome (FXS) is unclear. Our proteomic experiments reveal that the de novo translational profile in FXS model mice is altered at steady state and in response to metabotropic glutamate receptor (mGluR) stimulation, but the proteins expressed differ under these conditions. Several altered proteins, including Hexokinase 1 and Ras, also are expressed in the blood of FXS model mice and pharmacological treatments previously reported to ameliorate phenotypes modify their abundance in blood. In addition, plasma levels of Hexokinase 1 and Ras differ between FXS patients and healthy volunteers. Our data suggest that brain-based de novo proteomics in FXS model mice can be used to find altered expression of proteins in blood that could serve as disease-state biomarkers in individuals with FXS.

%B Nat Commun %V 10 %P 1710 %8 2019 Apr 12 %G eng %N 1 %R 10.1038/s41467-019-09553-8