Dataset Information

Optimization of protocols for detection of de novo protein synthesis in whole blood samples via azide-alkyne cycloaddition

ABSTRACT: Aberrant protein synthesis and protein expression are a hallmark of many disorders ranging from cancer Aberrant protein synthesis and protein expression are a hallmark of many disorders ranging from cancer to neuropsychiatry. 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 effort has been focused on unlabelled plasma proteomics which includes non-myeloid origin proteins and no method of dynamically tagging 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. Overall, we present a rapid and convenient de novo protein synthesis assay usable with whole blood biopsies that can quantify translational change as well as identity of proteins differentially expressed that may be useful for clinical applications

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Rattus Norvegicus (rat)

TISSUE(S): Blood Cell, Blood

SUBMITTER: Janani Priya Venkatasubramani

LAB HEAD: Dr. Aditi Bhattacharya

PROVIDER: PXD019243 | Pride | 2020-10-08

REPOSITORIES: Pride

ACCESS DATA

Publications

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

Bowling Heather L HL Kasper Amanda A Patole Chhaya C Venkatasubramani Janani Priya JP Leventer Sarah Parker SP Carmody Erin E Sharp Kevin K Berry-Kravis Elizabeth E Kirshenbaum Kent K Klann Eric E Bhattacharya Aditi A

Journal of proteome research 20200820 9

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 ...[more]

PMID: 32786687

Similar Datasets

Project description:To gain insight into the etiopathogenesis of Multiple sclerosis (MS) we investigated gene expression changes in CD4+ and CD8+ T lymphocytes from monozygotic twins (MZ) discordant for relapsing remitting MS. We studied 4 monozygotic twin pairs discordant for disease, with the affected co-twin free of disease modifying therapies (F/M = 3/1, mean age 36.25±3.9). Following leukapheresis, CD4+ and CD8+ T cells were separated and studied by Affymetrix GeneChip® For gene expression profiling, peripheral blood mononuclear cell (PBMC) were obtained from leukopheresis by density centrifugation over Ficoll-hypaque according to standard procedures. Briefly, heparinized blood was diluted with 1 volume of RPMI medium and gently layered over the Fycoll. Following centrifugation at 660 g for 30 minutes, cells at the interface of the gradient was collected and washed 3 times. Highly purified CD4+ and CD8+ (purity >95%) cell subsets were then sorted by flow cytometry on MoFlo high speed cell sorter (Beckman Coulter, Fullerton, CA) and frozen in Trizol (Invitrogen). Total RNA was extracted using TRIzol® (Invitrogen), according to the manufacturer's instructions. RNA quality and purity were assessed with the use of the RNA 6000 Nano assay on Agilent 2100 Bioanalyzer (Agilent). Total RNA from each sample was prepared using GeneChip® one Cycle cDNA Synthesis kit and GeneChip® IVT Labeling kit (Affymetrix), according to the manufacturer’s protocols. Briefly, starting from 2.5µg total RNA, biotinylated cRNA was produced by one cycle of reverse transcription and in vitro transcription (IVT). Biotinylated cRNA was fragmented and hybridized for 16 h at 45°C onto GeneChip® Human Genome U133 Plus 2.0 arrays (Affymetrix). The statistical analysis was performed couple by couple using the Rosetta Resolver® system (Rosetta Biosoftware). The fold-change of each gene was calculated by comparing gene expression in the affected twin with that in the unaffected twin and the genes with the same trend in at least 3 twin pairs out of the 4 studied were selected as candidates to be validated by real-time RT-PCR.

Dataset Information

Optimization of protocols for detection of de novo protein synthesis in whole blood samples via azide-alkyne cycloaddition

Publications

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

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure