Measurement of Protein Turnover Rates in Senescent and Non-Dividing Cultured Cells with Metabolic Labeling and Mass Spectrometry

Abstract

Mounting evidence has shown that the accumulation of senescent cells in the central nervous system contributes to neurodegenerative disorders such as Alzheimer's and Parkinson's diseases. Cellular senescence is a state of permanent cell cycle arrest that typically occurs in response to exposure to sub-lethal stresses. However, like other non-dividing cells, senescent cells remain metabolically active and carry out many functions that require unique transcriptional and translational demands and widespread changes in the intracellular and secreted proteomes. Understanding how protein synthesis and decay rates change during senescence can illuminate the underlying mechanisms of cellular senescence and find potential therapeutic avenues for diseases exacerbated by senescent cells. This paper describes a method for proteome-scale evaluation of protein half-lives in non-dividing cells using pulsed stable isotope labeling by amino acids in cell culture (pSILAC) in combination with mass spectrometry. pSILAC involves metabolic labeling of cells with stable heavy isotope-containing versions of amino acids. Coupled with modern mass spectrometry approaches, pSILAC enables the measurement of protein turnover of hundreds or thousands of proteins in complex mixtures. After metabolic labeling, the turnover dynamics of proteins can be determined based on the relative enrichment of heavy isotopes in peptides detected by mass spectrometry. In this protocol, a workflow is described for the generation of senescent fibroblast cultures and similarly arrested quiescent fibroblasts, as well as a simplified, single-time point pSILAC labeling time-course that maximizes coverage of anticipated protein turnover rates. Further, a pipeline is presented for the analysis of pSILAC mass spectrometry data and user-friendly calculation of protein degradation rates using spreadsheets. The application of this protocol can be extended beyond senescent cells to any non-dividing cultured cells such as neurons.

Figure 1:. Diagram of pSILAC workflow for senescent and quiescent control cells.

Human IMR-90 fibroblasts were used to prepare quiescent (low-serum) or senescent (IR) cell cultures for comparison of protein half-lives. Cells were then labeled with SILAC Light or SILAC Heavy media with isotopic arginine and lysine for 3 days. Lysates were extracted from cells, digested, desalted, and analyzed using mass spectrometry. Heavy and light peptide isotope peaks correspond to newly synthesized and pre-existing peptides, respectively. Half-lives were calculated using an equation for exponential decay.

Figure 2:. Validation of senescent phenotypes using SA-βGal and RT-qPCR analyses.

(A) Senescent and quiescent cells are re-plated at the time of harvest into a 6-well plate and stained for SA-βGal using the SA-βGal staining kit. Blue color in the cell body is positive for senescence. Images are taken in brightfield with color at 10x magnification, size marker in red. (B) RT-qPCR analysis comparing senescent cells (red) and cycling cells (gray) from an unrelated experiment. Increases in the levels of CDKN1A/p21, CXCL8, and IL6 mRNAs are indicative of senescence, as is the decrease in LMNB1 mRNA levels.

Figure 3:. Representative methods for data-dependent acquisition (DDA) scans and liquid chromatography gradient of pSILAC cultures on the Q-Exactive HF orbitrap mass spectrometer.

(A) Recommended instrument settings in the instrument software for data-dependent analysis of whole-cell lysates from a pSILAC experiment. (B) Example liquid chromatography flow gradient method settings. Peptides are eluted over a 90-min linear gradient ranging from 5% to 35% buffer B (0.2 % formic acid and 99.8% acetonitrile), followed by a 10 min wash with 80% buffer B, and 25 min of equilibration with 5% buffer B. (C) A representative total ion chromatogram (TIC) of a mass spectrometry acquisition of IMR-90 fibroblast peptides acquired with the specified settings.

Figure 4:. Representative extracted ion chromatograms of peptides with altered turnover during senescence.

(A) Chromatographic peak areas of the peptide FQMTQEVVCDECPNVK++ from the protein DnaJ homolog subfamily B member 11 (DNAJB11) in senescent and non-senescent cells. Following 3 days of SILAC, senescent cells incorporate less heavy isotope into this peptide compared with quiescent (non-senescent) cells, as indicated by a reduction in the peak area of heavy isotope-containing peptide (blue) relative to the light peptide (red), indicating that this peptide has reduced turnover in senescent cells. (B) Chromatographic peak areas of the peptide VQAQVIQETIVPK++ from the protein Splicing Factor 3a Subunit 1 (SF3A1) in senescent and non-senescent cells. Following 3 days of SILAC, senescent cells incorporate a higher proportion of heavy isotope into this peptide compared with quiescent (non-senescent) cells, as indicated by a reduction in the peak area of heavy isotope-containing peptide (blue) relative to the light peptide (red), indicating that this peptide has increased turnover in senescent cells. The unlabeled (Day 0) conditions show no incorporation of heavy isotope for both peptides, as expected.

Figure 5:. Comparison of protein half-lives in senescent and quiescent cells determined from pSILAC labeling.

(A) Volcano plot displaying the log2 ratio of senescent / control (quiescent) for each of the 695 identified proteins; in this experiment, Light and Heavy labeling media contained no glucose and no phenol red. (B) Tables showing the top 10 proteins with the most increased or decreased half-lives in quiescent cells versus senescent cells (left and right, respectively.