Time-resolved Analysis of Proteome Dynamics by Tandem Mass Tags and Stable Isotope Labeling in Cell Culture (TMT-SILAC) Hyperplexing

Abstract

Recent advances in mass spectrometry have enabled system-wide analyses of protein turnover. By globally quantifying the kinetics of protein clearance and synthesis, these methodologies can provide important insights into the regulation of the proteome under varying cellular and environmental conditions. To facilitate such analyses, we have employed a methodology that combines metabolic isotopic labeling (Stable Isotope Labeling in Cell Culture - SILAC) with isobaric tagging (Tandem Mass Tags - TMT) for analysis of multiplexed samples. The fractional labeling of multiple time-points can be measured in a single mass spectrometry run, providing temporally resolved measurements of protein turnover kinetics. To demonstrate the feasibility of the approach, we simultaneously measured the kinetics of protein clearance and accumulation for more than 3000 proteins in dividing and quiescent human fibroblasts and verified the accuracy of the measurements by comparison to established non-multiplexed approaches. The results indicate that upon reaching quiescence, fibroblasts compensate for lack of cellular growth by globally downregulating protein synthesis and upregulating protein degradation. The described methodology significantly reduces the cost and complexity of temporally-resolved dynamic proteomic experiments and improves the precision of proteome-wide turnover data.

Functional Translatome Proteomics Reveal Converging and Dose-Dependent Regulation by mTORC1 and eIF2α

“…In the last years, mass spectrometry (MS) approaches have helped in assessing protein dynamics by detecting protein degradation and synthesis, employing pulse-labeling of nascent peptide chains with heavy amino acid isotopes (SILAC) or clickreactive amino acids/puromycin (Becher et al, 2018;Jovanovic et al, 2015;Mathieson et al, 2018;Savitski et al, 2018;Schwanh€ ausser et al, 2009;Welle et al, 2016). A major limiting factor in the use of pulse-labeling newly synthesized proteins is the low stoichiometry of labeled proteins, preventing accurate, precise, and in-depth quantification (M€ unch and Harper, 2016).…”

“…A major limiting factor in the use of pulse-labeling newly synthesized proteins is the low stoichiometry of labeled proteins, preventing accurate, precise, and in-depth quantification (M€ unch and Harper, 2016). For basal protein degradation and synthesis experiments that monitor proteins over several days (Schwanh€ ausser et al, 2011), this issue has been overcome by combining pulse-labeling MS and tandem-mass tag (TMT)-based multiplexing (Welle et al, 2016). TMT allows isobaric tagging and pooling of up to 11 samples into one multiplexed sample.…”

Global impact of phosphorylation on protein endurance

“…1). In a typical pSILAC experiment, the growing cells are maintained in a steady-state in which the degraded and synthesized protein copies are balanced 28,29 . Such a concentration balance should also apply to most if not all modified proteinsotherwise, the system will be perturbed.…”

“…In a pSILAC experiment analyzing protein turnover, because the growing cells are respectively maintained in a steady state 29 , it is assumed that the degraded and synthesized protein copies are balanced 28 . Accordingly, almost all (if not all) the proteoforms, including alternative splicing isoforms 33 and proteins with PTMs, should achieve the concentration balance between synthesis and degradation in such a state (i.e., without any perturbation).…”

Allosteric HSP70 inhibitors perturb mitochondrial proteostasis and overcome proteasome inhibitor resistance in multiple myeloma

“…For these experiments we utilized JG342, to also evaluate if a similar phenotype is present across JG compounds. To distinguish effects on nascent vs. mature proteins and compare multiple timepoints in the same mass spectrometry experiment, we employed a modified version of recently-described methods of TMT combined with pulsed-SILAC proteomics 39,40 . Most commonly, SILAC (Stable Isotope Labeling of Amino acids in Cell culture) is utilized to compare the steady-state abundance of proteins under two different experimental conditions by incorporating different stable isotope-labeled amino acids resolvable by mass spectrometry 41 .…”