Proteoform Dynamics in Steady‐State

Abstract

Utilizing a classical dynamic SILAC (stable isotope labeling of amino acids in cell culture) approach, mass spectrometry‐based technologies nowadays allow for the determination of native protein turnover on a proteome‐wide scale. Obtaining proteoform resolution, however, is often still challenging due to incomplete quantitative information of peptides across pulse time‐points. To address this shortcoming, we evaluated the merits of (a) combining dynamic SILAC with TMT (tandem mass tag)‐labeling of ten pulse time‐points to compute absolute peptide turnover rates, and (b) utilizing solely single time‐point pulses to obtain relative differences in peptide half‐lives.In total, four cell culture replicates of each of the two different methodologies provided turnover information for 80,000 endogenously modified peptides assigned to up to 7,700 protein groups. Protein degradation rates were comparable to those reported with the standard pulsed SILAC approach. Moreover, replicate analyses established that the same reproducibility of turnover determination can be obtained for peptides and proteins eventually facilitating the investigation of proteoform resolved regulation of protein stability. Our comprehensive dataset showed that splice variants can exhibit vastly different cellular protein stabilities. Moreover, it revealed changes in turnover upon post‐translational modifications such as protein phosphorylation, ubiquitination and acetylation on a global scale. Several examples of differentially turned over peptides and proteins were investigated further. For example, differential peptide turnover indicated a previously unknown mechanism of activity regulation via post‐translational destabilization of only the light chain of cathepsin D as well as a phosphorylated version of DNA helicase BLM. Further, a parallel reaction monitoring assay applied to rotenone treated cells suggested that the high protein turnover of members of the respiratory chain complex I is affected by oxidative stress.Our data indicates that the original pulsed SILAC concept can be either expanded by employing TMT labeling of pulse time‐points to estimate absolute peptide turnover rates or simplified by using single time‐point pulses to evaluate relative differences in peptide half‐lives. Both strategies facilitated proteoform resolved turnover measurements. Collectively, they revealed a widely disregarded level of post‐transcriptionally and post‐translationally regulated protein stability which can have important implications for the study of disease mechanisms as well as the assessment of pharmacological interventions.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.