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

Abstract

SummaryTo 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 protein synthesis, with stronger down-regulation of glycolytic and protein synthesis machinery proteins, but up-regulation of several key chaperones. Waves of temporally distinct protein synthesis were observed in response to epidermal growth factor, with altered synthesis detectable in the first 15 min. Comparison of protein synthesis with mRNA sequencing and ribosome footprinting distinguished protein synthesis driven by increased transcription versus increased translational efficiency. Temporal delays between ribosome occupancy and protein synthesis were observed and found to correlate with altered codon usage in significantly delayed proteins.

One-shot analysis of translated mammalian lncRNAs with AHARIBO

“…2C). The lower number of proteins identified with AHARIBO-nP compared to pSILAC is most probably related to the shorter time of incubation with AHA (30 min) compared to pSILAC (24 hours) and is consistent with previous observations from similar pulldown enrichment strategies (Bagert et al, 2014; Rothenberg et al, 2018). Of note, 74 % of the AHARIBO-nP identified proteins is in common with the pSILAC dataset (Fig.…”

Loss of Elongator- and KEOPS-Dependent tRNA Modifications Leads to Severe Growth Phenotypes and Protein Aggregation in Yeast

“…While there is no clear bias in the use of t 6 A-dependent ANN codons in the loadings plot (brown circles in Figure 5B), there are several pairs of synonymous codon partners that strongly distinguish the up-and down-regulated proteins ( Figure 5B): ProCCG/down and ProCCA/up; SerTCC/down and SerAGC-AGT/up; HisCAC/down and HisCAT/up; AlaGCG/down and AlaGCT/up; ArgAGG/down and ArgAGA/up; IleATA/down and IleATT/ATC/up; GlyGGC/down and GlyGGT/up. We have observed this type of biased use of synonymous codon pairs in differentially regulated genes in yeast, bacteria, and human cells, with a strong link to coordinated changes in the tRNA pool and tRNA wobble modifications [77][78][79][80][81]. Codon usage in the 10 most up-and down-regulated proteins in the sua5 strain compared to wild-type was quantified using the codon utilization tool [82].…”

Systematic quantification of the dynamics of newly synthesized proteins unveiling their degradation pathways in human cells

“…7,8 However, most of the existing methods rely on antibodies and uorescence probes to investigate individual proteins, which limit their capacities for large-scale analysis of protein degradation. In recent years, mass spectrometry (MS)-based proteomics has provided an opportunity to comprehensively characterize proteins, [9][10][11][12][13][14][15][16][17][18][19][20][21] including protein dynamics. [22][23][24][25] For instance, Doherty et al studied the stability of nearly 600 proteins in human A549 cells by using a dynamic stable isotope labeling by amino acids in cell culture (SILAC) approach.…”

“…Half-life distributions of newly synthesized proteins in (a) different cellular compartments and protein complexes, (b) the core and regulatory subunits of the proteasome complex, (c) the scaffold and peripheral subunits in the nuclear pore complex, (d) the mitochondrial (mitochondrial ribosomal small subunit (MRS) and mitochondrial ribosomal large subunit (MRL)) and cytosolic (cytosolic ribosomal small subunit (RS) and cytosolic ribosomal large subunit (RL)) ribosome complexes.3560 | Chem. Sci., 2020,11,[3557][3558][3559][3560][3561][3562][3563][3564][3565][3566][3567][3568] This journal is © The Royal Society of Chemistry 2020Chemical Science Edge Article Open Access Article. Published on 10 March 2020.…”