Dataset Information

A proteomics approach to profiling the temporal translational response to stress and growth

ABSTRACT: One major knowledge gap in systems biology is the quantification of dynamic protein synthesis rates in response to cell perturbation. To address this gap, we have developed MITNCAT, multiplex isotope tagging / non-canonical amino acid tagging, a novel method enabling the robust quantification of proteome-wide protein synthesis rates with high temporal resolution (15-30 minutes) across multiple time points or treatment conditions, in a single analysis. MITNCAT combines multiplexed isobaric mass tagging with bioorthogonal non-canonical amino acid tagging (BONCAT) to label newly synthesized proteins with azidohomoalanine (Aha) and pulsed SILAC (pSILAC), thus providing enrichment and multiplexed quantification of newly synthesized proteins. We demonstrate the application of MITNCAT to quantify altered protein synthesis rates following induction of the unfolded protein response (UPR) and epidermal growth factor (EGF) stimulation. Eliciting the UPR by blocking N-glycosylation results in a global down-regulation of protein synthesis, with stronger down-regulation of proteins involved in the glycolysis pathway and protein synthesis machinery (ribosomal proteins, initiation factors, and elongation factors), but up-regulation of several key protein-folding chaperones. MITNCAT enables the quantification of waves of temporally distinct protein synthesis in response to EGF stimulation, with altered protein synthesis on dozens of proteins detectable in the first 15 minutes. Comparison of protein synthesis with RNA sequencing and ribosome footprinting allowed the distinction between protein synthesis driven by an increase in transcription versus that driven by an increase in translational efficiency. Temporal delays between ribosome occupancy and protein synthesis were observed and found to correlate with altered codon usage in significantly delayed proteins.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell, Cell Culture

SUBMITTER: Daniel Rothenberg

LAB HEAD: Forest White

PROVIDER: PXD009592 | Pride | 2018-12-04

REPOSITORIES: Pride

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Publications

A Proteomics Approach to Profiling the Temporal Translational Response to Stress and Growth.

Rothenberg Daniel A DA Taliaferro J Matthew JM Huber Sabrina M SM Begley Thomas J TJ Dedon Peter C PC White Forest M FM

iScience 20181105

To quantify dynamic protein synthesis rates, we developed MITNCAT, a method combining multiplexed isobaric mass tagging with pulsed SILAC (pSILAC) and bio-orthogonal non-canonical amino acid tagging (BONCAT) to label newly synthesized proteins with azidohomoalanine (Aha), thus enabling high temporal resolution across multiple conditions in a single analysis. MITNCAT quantification of protein synthesis rates following induction of the unfolded protein response revealed global down-regulation of p ...[more]

PMID: 30466063

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Project description:Food-borne illness arising for Shiga-toxigenic Escherichia coli is often linked to consumption of fruit and vegetables as the bacteria have the ability to interact with plants and use them as alternative or secondary hosts. Attachment of the bacteria to host tissue is one of the first steps in the interaction, and, as with mammalian hosts, has shown to be mediated by a combination of non-specific and specific adhesin-mediated interactions. We took a high-throughput positive-selection approach to investigate adherence mechanisms for E. coli O157:H7 isolate Sakai by inoculating a BAC clone library onto spinach, which was quantified by microarray hybridisation and gene loci enrichment measured using a Bayesian hierarchical model. The screen involved four successive rounds of adherence to spinach roots, resulting in 115 CDS credible candidates, covered by seven contiguous genomic regions. Two candidates regions selected for functional assessment included a chaperone-usher fimbrial gene cluster (loc6) and the type two secretion system (T2SS). The TS22 was found to significantly enhance binding to spinach roots and leaves, demonstrated with a BAC-T2SS clone and by mutagenesis of the secretin protein, EtpD. Both etpD and the inner membrane anchor protein gene etpC were expressed at 18 degree celsius, and expression of etpD was demonstrated for STEC (Sakai) resident in the apoplastic spaces in spinach leaf tissue. Together, these data indicate a novel function for STEC T2SS in adherence to plant tissue. Experiment 1: screening E coli O157:H7 Sakai genes for adherence to spinach roots. A BAC library of Sakai clones in an E coli DH10B background (which has poor root adherence) defined as the 'Input pool', was incubated with spinach roots for 4 rounds of enrichment, defined as the 'Output pool'. Control samples (defined as 'Input control' & 'Output control') were cultures of pV41 vector only. DNA extractions from test pools were labelled with Cy3 throughout. DH10B DNA was used for grid alignment and labelled with Cy5 throughout.

Dataset Information

A proteomics approach to profiling the temporal translational response to stress and growth

Publications

A Proteomics Approach to Profiling the Temporal Translational Response to Stress and Growth.

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