This work was supported by the National Institutes of Health (NIH) and the Intramural Research Program (IRP), National Institute on Aging (NIA). N.B. was supported by Longevity Impetus Grants, and the Office of Dietary Supplements (ODS) Scholars Program. Figure 1 was created with BioRender.com.

The authors have no disclosures.

pSILAC is a powerful technique that enables the global quantification of protein turnover rates across multiple cellular conditions. This paper details the use of pSILAC to compare global protein half-lives between senescent and quiescent cells, including instructions for the preparation of senescent and quiescent cells, SILAC labeling and harvesting, and ultimately analysis using DDA mass spectrometry. Additionally, a two-step test is described for validation of the senescence phenotype using SA-&#946;Gal and RT-qPCR analysis of a panel of mRNAs encoding senescence-associated proteins. In addition to validation of senescence with the two approaches described, a third validation of senescence can be performed following mass spectrometry analysis by looking for changes in known senescence markers between senescent and quiescent cells at the proteomic level. Senescence-associated proteins that are expected to be elevated include p16, p21, and BCL2, among others, described elsewhere44,45. In the protocol described above, ionizing radiation was used for the induction of senescence and serum starvation for quiescent cells. For the induction of senescence, there are multiple options available and there is substantial heterogeneity among them41,42,46. Currently, there is no senescent method that is considered the &#34;most physiological&#34;, so the choice of senescent inducer is largely based on the context of the experiment. However, experiments with the goal of stating a general phenomenon about senescence are recommended to use at least two different senescent inducers. Discussing the range of senescence paradigms is beyond the scope of this paper, but some common methods to induce senescence include triggering DNA damage (IR, doxorubicin, replicative exhaustion), expressing oncogenic proteins (HRAS, BRAF), and disrupting mitochondria function2.
In addition to the choice of senescent inducer, the choice of control cells is an equally important consideration. Senescent cells are, by definition, under indefinite growth arrest, so a comparison to other growth-arrested cells are often chosen. For pSILAC, cell cycle arrested cells are generally preferable because they do not replicate and are thus easier to use for protein half-life calculations47. However, since cultured cells will often retain some dividing cells, it is important that the methods used to induce cell cycle arrest produce as homogenous a response as possible to minimize error from cells that are still proliferating. To calculate protein degradation rates for cycling cells using pSILAC requires additional calculations to compensate for the rate at which protein is diluted into daughter cells27. However, quiescent growth arrest itself is not without complications. There are two general methods for cell cycle arrest: serum deprivation and contact inhibition48. Not all cells can be made quiescent through contact inhibition, although some fibroblasts have been shown to demonstrate quiescence after several days of culturing49. This method used serum deprivation because it is more commonly used for comparisons of senescent cells, though it requires for the senescent cell to be similarly serum-deprived for accurate comparisons. Serum activates the mTOR complex, and thus serum deprivation has several downstream effects on the cell in addition to cell cycle arrest50. Notably, senescent cells have been shown to display a reduced SASP upon serum deprivation or mTOR inhibition51,52.
Another important point to consider in pSILAC is how many time points to test. This protocol collected cells at a single time point (3 days of light or heavy labeling), which substantially simplifies the resulting analysis. The choice of time point should be based on the objective of the experiment. For global analysis, 3 days is expected to capture a majority of proteins, though half-lives for short-lived proteins that completely turnover within 3 days (all light signal is lost) cannot be measured at this time point. Conversely, long-lived proteins with very little turnover in 3 days are also difficult to quantify and often appear as having extremely large half-lives (in the order of weeks) that are typically just a consequence of very little heavy signal accumulation. Due to the non-linear relationship in the ratio of heavy and light peptide signals versus the percentage of newly synthesized protein at shorter and longer time points, the quantitation of half-lives could be improved by adding additional labeling time points. For relative comparisons between two cell states, as in this protocol, an approximate half-life may be sufficient, but additional timepoints can be used to improve quantitative accuracy.
This protocol describes how to perform an untargeted DDA-based analysis of protein turnover. However, the protein turnover calculations can be applied generally to any acquisition scheme that is able to derive the relative abundance of heavy and light peptide pairs. For example, MS2-based methods such as data-independent acquisition (DIA/SWATH) can also be applied for the calculation of turnover rates successfully53. Additionally, instrumentation and software pipelines other than those described in this protocol can be used to perform DDA analysis, protein identification, and protein quantification. When using protein quantification software platforms such as Skyline to extract peptide peak areas, it is advisable to manually inspect extracted ion chromatograms in the document workspace, identify peaks that were erroneously integrated and non-quantitative peaks, and curate the document accordingly. An extensive collection of tutorials is available online for Skyline (skyline.ms).
pSILAC represents one of the most ideal methods for global quantification of protein half-lives in cultured cells due to superior multiplexing (proteome coverage) and throughput. While pSILAC does not provide direct rates of synthesis or degradation, since the change in light and heavy signal is due to a confluence of factors, pSILAC is highly useful for comparisons between conditions and different cell types. Low-throughput methods often fall into two types: 1) treatment of cells with cycloheximide to block protein synthesis and harvest at time intervals after addition to monitor decay, or 2) treatment of cells with an inhibitor of protein decay and harvest at time intervals after addition to monitor the accumulation of protein, thus inferring protein decay rates. The limitation of both methods is that such treatments will inevitably cause substantial changes to cellular physiology. In contrast, pSILAC requires no substantial intervention and theoretically has no detectable effects on cellular physiology since isotopic amino acids differ by only a single neutron from their non-isotopic counterparts. Thus, the method described here for pSILAC represents a simple protocol for global measurement of the most physiological protein half-lives in non-dividing cells.
Alterations in protein turnover have a close relationship to aging, age-related diseases, neurodegeneration, and longevity54,55. This protocol describes a method to interrogate these relationships by using stable isotope labeling of amino acids in cell culture to measure protein turnover rates in senescence cells. However, numerous analogous methods exist to perform studies in the context of aging and neurodegeneration in vivo in whole organisms such as mice. Indeed, these studies have emphasized the importance of measuring protein turnover rates in the context of age-related diseases56,57,58,59.
In this study, ribosomal proteins and proteins residing in the endoplasmic reticulum&#160;stood out as two categories of proteins with decreased and increased half-lives in senescent cells, respectively. While further analysis of steady-state levels is required for definitive conclusions, these results further suggest that senescent cells may uniquely regulate translation through decreased half-lives of ribosomal proteins. Going forward, applying stable isotope labeling approaches to study the relationship between cellular senescence and neurodegeneration in vivo in mouse models will be a promising extension of the isotope labeling approach described by this protocol.

Senescence was first identified as a state of indefinite growth arrest exhibited by cultured primary cells after reaching replicative exhaustion1. It has since been shown that senescence can arise in response to numerous cellular insults, including genotoxic, mitochondrial, and oncogenic stresses, among others2. While senescence has several physiologically important roles, such as tumor suppression and wound healing, the accumulation of senescent cells during aging is associated with a host of deleterious effects on health3, including several neurodegenerative conditions4,5,6. Cellular senescence occurs in multiple brain cell types, including neurons7,8,9,10, astrocytes11, microglia12, and oligodendrocyte precursors13, and contributes to neurodegeneration and cognitive dysfunction. Amyloid beta oligomers, one of the hallmarks of Alzheimer&#39;s disease14, have been shown to accelerate neuronal senescence13,15,16. An increased prevalence of senescent cells has also been associated with Parkinson&#39;s disease17, especially arising from environmental stressors11,18. Importantly, the selective elimination of senescent cells in pre-clinical models extends lifespan and mitigates a multitude of age-related diseases3,5,12 and improves cognitive deficits8,11,12,13. Senescent cells have thus emerged as promising therapeutic targets for the treatment of many age-related conditions.
Much of the detrimental effect of senescent cells is caused by the senescence-associated secretory phenotype (SASP), a complex mixture of bioactive molecules secreted by senescent cells that can cause local inflammation, angiogenesis, destruction of the extracellular matrix, and propagation of senescence in surrounding tissue19,20,21. The SASP also represents an interesting biological phenomenon of senescence because it requires a considerable transcriptional and translational effort during a state of cell cycle arrest. In fact, senescent cells have been shown to exhibit decreases in ribosome biogenesis22,23,24 that should reduce protein synthesis. Instead, senescent cells robustly translate some proteins, particularly SASP factors, and influence the metabolism of surrounding tissue25. Thus, there is considerable interest in understanding how senescent cells undergoing a permanent cell cycle arrest continue to maintain protein homeostasis while at the same time robustly express SASP factors and other select proteins.
This method describes how to use mass spectrometry and pulsed-stable isotope labeling by amino acids in cell culture (pSILAC) to globally measure the half-lives of proteins in senescent cells at a proteome-wide scale. In traditional SILAC, cultured cells are completely metabolically labeled with heavy and light non-radioactive isotopes of amino acids for downstream analysis of protein abundance. This method has been previously applied to assess abundance changes comprehensively and quantitatively in the SASP of cultured fibroblasts26. In pSILAC, cells are similarly metabolically labeled with a pulse of heavy isotope that follows pre-labeling with light isotope, and then harvested at one or more time intervals. The rates of incorporation of heavy isotope in reference to pre-existing light isotope are then used to calculate relative protein turnover rates. Generally, isotopes of arginine and lysine are used because trypsin cleaves at those residues; thus, all peptides from standard digestion will potentially contain the heavy label. Pairs of peptides that differ only by the presence or absence of heavy lysine or arginine are chemically identical and can be differentiated and quantified by a mass spectrometer. Following mass spectrometry analysis, peptides can be identified as newly synthesized or pre-existing based on the presence or absence of the isotopic label in the resulting peptide identifications. Protein turnover rates can then be determined by fitting the ratio of heavy (13C-15N) over light (12C-14N) peptides for a given protein to kinetic models for exponential growth or decay27,28. pSILAC has been used in several comparisons of protein turnover rates29,30,31,32 and is currently the most comprehensive and high-throughput method for the measurement of protein half-lives.
This protocol details the preparation of senescent cells in parallel with similarly growth-arrested quiescent cells in culture, followed by metabolic labeling with pSILAC. Cells are then harvested, homogenized into lysates, and processed for mass spectrometry acquisition and analysis. The data obtained from mass spectrometry are then used to determine protein half-lives using a simplified quantitative method employing a single time point and half-life calculations performed in a spreadsheet. Using this approach, estimates of protein half-lives can be measured in a comprehensive and quantitative manner that is more authentic to unperturbed cellular conditions than protocols that use blockers of protein synthesis or turnover.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Acetonitrile (LC-MS grade)</td>
			<td>Grainger</td>
			<td>AH015</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium Bicarbonate</td>
			<td>Millipore-Sigma</td>
			<td>9830</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic (100x)</td>
			<td>ThermoFisher</td>
			<td>15240062</td>
			<td></td>
		</tr>
		<tr>
			<td>BCA Assay Kit</td>
			<td>ThermoFisher</td>
			<td>23227</td>
			<td></td>
		</tr>
		<tr>
			<td>Dithiothreotol (DTT)</td>
			<td>Sigma</td>
			<td>D9779</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM, high glucose, HEPES</td>
			<td>ThermoFisher</td>
			<td>12430112</td>
			<td></td>
		</tr>
		<tr>
			<td>dNTP Mix</td>
			<td>ThermoFisher</td>
			<td>R0191</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum, certified, heat inactivated</td>
			<td>ThermoFisher</td>
			<td>10082147</td>
			<td></td>
		</tr>
		<tr>
			<td>Formic Acid</td>
			<td>Sigma</td>
			<td>27001</td>
			<td></td>
		</tr>
		<tr>
			<td>Gammacel 40 Exactor</td>
			<td>Best Theratronics</td>
			<td></td>
			<td>Cesium Irradiator for cells</td>
		</tr>
		<tr>
			<td>GlycoBlue</td>
			<td>ThermoFisher</td>
			<td>AM2238</td>
			<td></td>
		</tr>
		<tr>
			<td>Iodoacetamide (IAA)</td>
			<td>Sigma</td>
			<td>I1149</td>
			<td>Light sensitive</td>
		</tr>
		<tr>
			<td>IMR-90 primary lung fibroblasts</td>
			<td>ATCC</td>
			<td>CCL-186</td>
			<td></td>
		</tr>
		<tr>
			<td>iRT Kit (indexed retention time)</td>
			<td>Biognosys</td>
			<td>Ki-3002-2</td>
			<td>Indexed Retention Time Peptide Standards</td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>ThermoFisher</td>
			<td>423835000</td>
			<td></td>
		</tr>
		<tr>
			<td>Mascot</td>
			<td>Matrix Science</td>
			<td>Mascot Daemon 2.8</td>
			<td>Proteomic database searching software</td>
		</tr>
		<tr>
			<td>Maxima Reverse Transcritase (200 U/&#181;L)</td>
			<td>ThermoFisher</td>
			<td>EP0742</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM Non-Essential Amino Acids Solution (100x)</td>
			<td>ThermoFisher</td>
			<td>11140050</td>
			<td></td>
		</tr>
		<tr>
			<td>Nano LC System</td>
			<td>ThermoFisher</td>
			<td>ULTIM3000RSLCNANO</td>
			<td></td>
		</tr>
		<tr>
			<td>Oasis HLB Solid Phase Extraction Cartirdges</td>
			<td>Waters</td>
			<td>186000383</td>
			<td></td>
		</tr>
		<tr>
			<td>Orbitrap Mass Spectrometer</td>
			<td>ThermoFisher</td>
			<td>Q Exactive HF Orbitrap</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenol/Chloroform/Isoamyl alcohol (25:24:1), 100 mM EDTA, pH 8.0</td>
			<td>ThermoFisher</td>
			<td>327110025</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS)</td>
			<td>ThermoFisher</td>
			<td>10010023</td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce SILAC Protein Quantitation Kit (Trypsin) -DMEM</td>
			<td>ThermoFisher</td>
			<td>A33972</td>
			<td></td>
		</tr>
		<tr>
			<td>QuantStudio 6 Real-Time PCR System</td>
			<td>ThermoFisher</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Random Hexamer Primer</td>
			<td>ThermoFisher</td>
			<td>SO142</td>
			<td></td>
		</tr>
		<tr>
			<td>Senescence &#946;-Galactosidase Staining Kit</td>
			<td>Cell Signaling</td>
			<td>9860</td>
			<td></td>
		</tr>
		<tr>
			<td>Skyline</td>
			<td>University of Washington</td>
			<td>Skyline-Daily v21.2.1.424</td>
			<td>Free and open source qantiative proteomic software. Available on www.skyline.ms</td>
		</tr>
		<tr>
			<td>Sonicator waterbath</td>
			<td>Branson</td>
			<td>CPX-952-516R</td>
			<td></td>
		</tr>
		<tr>
			<td>TRIzol Reagent</td>
			<td>ThermoFisher</td>
			<td>15596018</td>
			<td>Referred to as phenol in text; hazardous</td>
		</tr>
		<tr>
			<td>TRYPle Express</td>
			<td>ThermoFisher</td>
			<td>12605010</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin (sequencing grade)</td>
			<td>Promega</td>
			<td>V5113</td>
			<td></td>
		</tr>
		<tr>
			<td>TURBO Dnase (2U/ uL)</td>
			<td>ThermoFisher</td>
			<td>AM2238</td>
			<td></td>
		</tr>
		<tr>
			<td>Urea</td>
			<td>ThermoFisher</td>
			<td>29700</td>
			<td></td>
		</tr>
		<tr>
			<td>Water (LC-MS grade)</td>
			<td>Grainger</td>
			<td>AH365</td>
			<td></td>
		</tr>
	</tbody>
</table>

measurement of protein turnover rates in senescent and non-dividing cultured cells with metabolic labeling and mass spectrometry

Mounting evidence has shown that the accumulation of senescent cells in the central nervous system contributes to neurodegenerative disorders such as Alzheimer&#39;s and Parkinson&#39;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.

This protocol describes a method to globally compare protein half-lives between senescent and non-dividing, quiescent control cells using pSILAC and minimal time points. This protocol details generation of senescent and quiescent cells in culture, metabolic labeling of cells with stable isotopes of arginine and lysine over 3 days, quantifying the relative abundances of heavy and light peptide isotopes by mass spectrometry, and a straightforward and accessible calculation of protein half-lives using spreadsheet formulas (Figure 1). This method is highly flexible and can be adapted to numerous cell types and conditions.
As part of the generation of senescent cells for this protocol, two methods of senescence validation are used: SA-&#946;Gal-positive cells visualized by microscopy and increased levels of senescent markers quantified using RT-qPCR analysis. The measurement of senescent markers should yield clear distinctions between quiescent and senescent cells for comparisons of the two cells populations to be considered valid. For SA-&#946;Gal activity, senescent cells should appear blue while quiescent control cells have no or very little color (Figure 2A). This assay can be quantified by counting the positive, blue-stained cells as a percentage of the total number of cells, and then comparing the percentage positivity rate between quiescent control and senescent cells. It is important that this protocol be performed at the same time for the comparison of both cell states; SA-&#946;Gal activity relies on the pH of the staining solution, so the results can vary substantially between assays and should be considered a qualitative measure.
The RT-qPCR analysis of senescent markers will show high levels of the mRNAs encoding SASP factors (IL6, CXCL8, IL1B) and the cell cycle inhibitors (CDKN2A/p16, CDKN1A/p21) in most senescent models. Although some variation is expected based on cell type, culture condition, and senescent inducer41,42, most senescent cells display greater than five-fold higher levels of IL6, CXCL8, and CDKN1A/p21 mRNAs compared to quiescent control cells; conversely, the levels of LMNB1 and PCNA mRNAs, encoding proliferation markers, should be low or absent in senescent cells compared with quiescent&#160;cells (Figure 2B). Taken together, a significant percentage of SA-&#946;Gal positivity and the expected expression of a panel of senescence-associated mRNA markers are sufficient to confirm the induction of senescence in an experiment.
Processing protein samples for mass spectrometry analysis consists of in-solution digestion (2 days) and solid-phase extraction (4-6 h). To perform untargeted proteomic analysis, the resulting peptides are submitted for LC-MS/MS analysis using data-dependent acquisition (DDA). On Orbitrap instruments, a DDA method can be specified in the mass spectrometry instrument software method editor. The mass spectrometer settings used in this study are shown in Figure 3A. Liquid chromatography settings can also be specified within the method editor. For this study, a 90 min linear gradient with increasing organic phase (acetonitrile) was used (Figure 3B). Following a successful acquisition, the total ion current (TIC) should contain an intense signal during the linear gradient portion of the method (Figure 3C). This protocol describes an example protocol on an Orbitrap instrument, but the calculation of protein half-lives can be performed on data obtained from any mass spectrometer that was collected in DDA-mode, which is available on several types of instruments (e.g., Orbitraps and time-of-flight instruments) and vendors. The acquisition settings will be unique to the type and configuration of the instrument used, and it is recommended to use the settings suggested by the mass spectrometry facility. This method is also compatible with non-DDA methods (SRM, PRM, DIA), as long as quantitative chromatographic peak areas for heavy and light peaks can be extracted from the raw data files and entered into the spreadsheet formulas.
Raw mass spectrometry files are searched with one of the many available proteomic database search tools to identify peptides and proteins. For example, this study utilized the Mascot43 search engine. To obtain chromatographic peak areas for quantification of heavy and light peptides, database search results are imported into a proteomic software capable of chromatographic peak areas such as Skyline39,40. Examination of the extracted ion chromatograms of peptides (Figure 4) will reveal the relative proportion of heavy and light peptide signals. A lower proportion of heavy peptide signal relative to light peptide signal in senescent cells indicates a slower protein turnover rate (Figure 4A), and a higher heavy peptide signal relative to light indicates a faster protein turnover rate (Figure 4B). The unlabeled samples should show little or no heavy peptide signal. Any apparent heavy peptide signal in the unlabeled samples is considered background noise and will be subtracted during the final calculations. Quantification of chromatographic peak areas for light and heavy peptides for all treatment and labeling conditions must be exported for subsequent calculation of protein turnover rates and statistical analysis.
By using single time point analysis, the quantification of protein half-lives is simple to perform and can be done conveniently in spreadsheets. In Table 3, the half-lives for 695 proteins identified from the mass spectrometry analysis were calculated. Starting from protein-level heavy and light isotope abundances for each protein in the samples (input for the 1) Raw Data sheet), a percent heavy isotope (termed the Ratio, R) is then automatically calculated on the sheet titled 2) Ratio H | H + L. At this step, the R from heavy labeled samples is normalized by subtracting the R from unlabeled samples to produce a final R for the quiescent and senescent triplicates. Since unlabeled samples should not have any exogenously added heavy isotope, they are used to eliminate the background signal. From R, the kdeg (turnover rate) is calculated using the following formula:
<img alt="Equation 1" src="/files/ftp_upload/63835/63835eq01.jpg" style="margin: auto;" />
The half-life (H, in days) is determined for each protein in the quiescent control and senescent triplicates (sheet titled 3) Half-Life (days)) using the following formula:
<img alt="Equation 2" src="/files/ftp_upload/63835/63835eq02.jpg" style="margin: auto;" />
These half-lives are then averaged among the quiescent and senescent triplicates to generate a quiescent and senescent mean half-life for each protein as well as a p-value. Calculated half-lives are then filtered for values that are negative, which occurs when the heavy signal from light-labeled cells (the background) is greater than the heavy signal from heavy-labeled cells. This filtering resulted in 707 proteins from 841 identified with valid half-lives for the results presented here.
The half-life is then reported as a log2 ratio of senescent over quiescent control cells (log2FC) and plotted in a volcano plot (Figure 5). For example, by looking at the 5) Analysis sheet, the coagulation II thrombin receptor (F2R) protein can be seen to have a half-life in quiescent cells of 0.51 days (column B) and a half-life in senescent cells of 1.07 days (column C) that yielded a log2FC of ~1.06 (column D) with a p-value of 0.001.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63835/63835fig01.jpg" /> 
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. <a href="https://www.jove.com/files/ftp_upload/63835/63835fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63835/63835fig02.jpg" /> 
Figure 2: Validation of senescent phenotypes using SA-&#946;Gal 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-&#946;Gal using the SA-&#946;Gal 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. <a href="https://www.jove.com/files/ftp_upload/63835/63835fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63835/63835fig03.jpg" /> 
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. <a href="https://www.jove.com/files/ftp_upload/63835/63835fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63835/63835fig04.jpg" /> 
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. <a href="https://www.jove.com/files/ftp_upload/63835/63835fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/63835/63835fig05.jpg" /> 
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). <a href="https://www.jove.com/files/ftp_upload/63835/63835fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1: Media and buffers used in this protocol. <a href="https://www.jove.com/files/ftp_upload/63835/Table 1-63835R1.xlsx" target="_blank">Please click here to download this Table.</a>
Table 2: RT-qPCR primers used in this protocol. <a href="https://www.jove.com/files/ftp_upload/63835/Table 2-63835R1.xlsx" target="_blank">Please click here to download this Table.</a>
Table 3: SILAC analysis workbook for calculation of protein half-lives, fold changes, and t-tests. <a href="https://www.jove.com/files/ftp_upload/63835/Table 3 v3.xlsx" target="_blank">Please click here to download this Table.</a>

Watch this Scientific Journal Video about Measurement of Protein Turnover Rates in Senescent and Non-Dividing Cultured Cells with Metabolic Labeling and Mass Spectrometry at JoVE.com

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

This protocol describes the workflow for metabolic labeling of senescent and non-dividing cells with pulsed SILAC, untargeted mass spectrometry analysis, and a streamlined calculation of protein half-lives.

Mounting evidence has shown that the accumulation of senescent cells in the central nervous system contributes to neurodegenerative disorders such as ...

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JoVE Journal

Biochemistry

3.3K Views.  National Institutes of Health. This protocol describes the workflow for metabolic labeling of senescent and non-dividing cells with pulsed SILAC, untargeted mass spectrometry analysis, and a streamlined calculation of protein half-lives.

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

Sheathless Capillary Electrophoresis&#8211;Mass Spectrometry for Metabolic Profiling of Biological Samples

In metabolomics, a wide range of analytical techniques is used for the global profiling of (endogenous) metabolites in complex samples. In this paper, a protocol is presented for the analysis of anionic and cationic metabolites in biological samples by capillary electrophoresis&#8211;mass spectrometry (CE-MS). CE is well-suited for the analysis of highly polar and charged metabolites as compounds are separated on the basis of their charge-to-size ratio. A recently developed sheathless interfacing design, i.e., a porous tip interface, is used for coupling CE to electrospray ionization (ESI) MS. This interfacing approach allows the effective use of the intrinsically low-flow property of CE in combination with MS, resulting in nanomolar detection limits for a broad range of polar metabolite classes. The protocol presented here is based on employing a bare fused-silica capillary with a porous tip emitter at low-pH separation conditions for the analysis of a broad array of metabolite classes in biological samples. It is demonstrated that the same sheathless CE-MS method can be used for the profiling of cationic metabolites, including amino acids, nucleosides and small peptides, or anionic metabolites, including sugar phosphates, nucleotides and organic acids, by only switching the MS detection and separation voltage polarity. Highly information-rich metabolic profiles in various biological samples, such as urine, cerebrospinal fluid and extracts of the glioblastoma cell line, can be obtained by this protocol in less than 1 hr of CE-MS analysis.

A protocol for metabolic profiling of biological samples by capillary electrophoresis&#8211;mass spectrometry using a sheathless porous tip interface design is presented.

In metabolomics, a wide range of analytical techniques is used for the global profiling of (endogenous) metabolites in complex samples. In this paper, a ...

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. Hydrogen-Deuterium Exchange (HDX) measured at rapid time scales is uniquely suited to detect structures and hydrogen bonding networks that are briefly populated, allowing for the characterization of transient conformers in native ensembles. Coupling of HDX to mass spectrometry offers several key advantages, including high sensitivity, low sample consumption and no restriction on protein size. This technique has advanced greatly in the last several decades, including the ability to monitor HDX labeling times on the millisecond time scale. In addition, by incorporating the HDX workflow onto a microfluidic platform housing an acidic protease microreactor, we are able to localize dynamic properties at the peptide level. In this study, Time-Resolved ElectroSpray Ionization Mass Spectrometry (TRESI-MS) coupled to HDX was used to provide a detailed picture of residual structure in the tau protein, as well as the conformational shifts induced upon hyperphosphorylation.

Conformational flexibility plays a critical role in protein function. Herein, we describe the use of time-resolved electrospray ionization mass spectrometry coupled to hydrogen-deuterium exchange for probing the rapid structural changes that drive function in ordered and disordered proteins.

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

Resolving Affinity Purified Protein Complexes by Blue Native PAGE and Protein Correlation Profiling

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological processes. Affinity purification coupled to mass spectrometry (AP-MS) has become the method of choice for identifying protein-protein interactions. However, conventional AP-MS studies provide information on protein interactions, but the organizational information is lost. To address this issue, we developed a strategy to unravel the distinct functional assemblies a protein might be involved in, by resolving affinity-purified protein complexes prior to their characterization by mass spectrometry. Protein complexes isolated through affinity purification of a bait protein using an epitope tag and competitive elution are separated through blue native electrophoresis. Comparison of protein migration profiles through correlation profiling using quantitative mass spectrometry allows assignment of interacting proteins to distinct molecular entities. This method is able to resolve protein complexes of close molecular weights that might not be resolved by traditional chromatographic techniques such as gel filtration. With little more work than conventional AP-geLC-MS/MS, we demonstrate this strategy may in many cases be adequate for obtaining protein complex topological information concomitantly to identifying protein interactions.

Here we present protocols for affinity purification of protein complexes and their separation by blue native PAGE, followed by protein correlation profiling using label free quantitative mass spectrometry. This method is useful to resolve interactomes into distinct protein complexes.

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological ...

Mass Spectrometry and Luminogenic-based Approaches to Characterize Phase I Metabolic Competency of In Vitro Cell Cultures

Xenobiotic metabolizing enzymes play a key function in the biotransformation of medicines and toxicants by adding functional groups that increase solubility and facilitate excretion. On some occasions those structural modifications lead to the formation of new toxic products. In order to reduce animal testing, chemical risk can be assessed using metabolically competent cells. The expression of metabolic enzymes, however, is not stable over time in many in vitro primary culture systems and is often partial or absent in cell lines. Therefore, the study of medicines, additives, and environmental pollutants metabolism in vitro should ideally be conducted in cell systems where metabolic activity has been characterized. We explain here an approach to measure the activity of a class of metabolic enzymes (Human Phase I) in 2D cell lines and primary 3D cultures using chemical probes and their metabolic products quantifiable by UPLC mass spectrometry and luminometry. The method can be implemented to test the metabolic activity in cell lines and primary cells derived from a variety of tissues.

Mass Spectrometry and Luminogenic-based Approaches to Characterize Phase I Metabolic Competency of In Vitro Cell Cultures

Metabolic competency of in vitro systems is a key requirement for the biotransformation and disposition of drugs and toxicants. In this protocol, we describe the application of reference metabolic probes to assess phase I metabolism in cell cultures.

Xenobiotic metabolizing enzymes play a key function in the biotransformation of medicines and toxicants by adding functional groups that increase ...

Sample Preparation for Mass Spectrometry-based Identification of RNA-binding Regions

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most cases, the action of noncoding RNAs is mediated by proteins whose functions are in turn regulated by these interactions with noncoding RNAs. Consistent with this, a growing number of proteins involved in nuclear functions have been reported to bind RNA and in a few cases the RNA-binding regions of these proteins have been mapped, often through laborious, candidate-based methods.
Here, we report a detailed protocol to perform a high-throughput, proteome-wide unbiased identification of RNA-binding proteins and their RNA-binding regions. The methodology relies on the incorporation of a photoreactive uridine analog in the cellular RNA, followed by UV-mediated protein-RNA crosslinking, and mass spectrometry analyses to reveal RNA-crosslinked peptides within the proteome. Although we describe the procedure for mouse embryonic stem cells, the protocol should be easily adapted to a variety of cultured cells.

We describe a protocol to identify RNA-binding proteins and map their RNA-binding regions in live cells using UV-mediated photocrosslinking and mass spectrometry.

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most ...

Deep Proteome Profiling by Isobaric Labeling, Extensive Liquid Chromatography, Mass Spectrometry, and Software-assisted Quantification

Many exceptional advances have been made in mass spectrometry (MS)-based proteomics, with particular technical progress in liquid chromatography (LC) coupled to tandem mass spectrometry (LC-MS/MS) and isobaric labeling multiplexing capacity. Here, we introduce a deep-proteomics profiling protocol that combines 10-plex tandem mass tag (TMT) labeling with an extensive LC/LC-MS/MS platform, and post-MS computational interference correction to accurately quantitate whole proteomes. This protocol includes the following main steps: protein extraction and digestion, TMT labeling, 2-dimensional (2D) LC, high-resolution mass spectrometry, and computational data processing. Quality control steps are included for troubleshooting and evaluating experimental variation. More than 10,000 proteins in mammalian samples can be confidently quantitated with this protocol. This protocol can also be applied to the quantitation of post translational modifications with minor changes. This multiplexed, robust method provides a powerful tool for proteomic analysis in a variety of complex samples, including cell culture, animal tissues, and human clinical specimens.

We present a protocol to accurately quantitate proteins with isobaric labelling, extensive fractionation, bioinformatics tools, and quality control steps in combination with liquid chromatography interfaced to a high-resolution mass spectrometer.

Many exceptional advances have been made in mass spectrometry (MS)-based proteomics, with particular technical progress in liquid chromatography (LC) ...

Measuring and Interpreting Oxygen Consumption Rates in Whole Fly Head Segments

Regulated metabolic activity is essential for the normal functioning of living cells. Indeed, altered metabolic activity is causally linked with the progression of cancer, diabetes, neurodegeneration, and aging to name a few. For instance, changes in mitochondrial activity, the cell&#39;s metabolic powerhouse, have been characterized in many such diseases. Generally, the oxygen consumption rates of mitochondria were considered a reliable readout of mitochondrial activity and measurements in some of these studies were based on isolated mitochondria or cells. However, such conditions may not represent the complexity of a whole tissue. Recently, we have developed a novel method that enables the dynamic measurement of oxygen consumption rates from whole isolated fly heads. By utilizing this method, we have recorded lower oxygen consumption rates of the whole head segment in young versus aged flies. Secondly, we have discovered that lysine deacetylase inhibitors rapidly alter the oxygen consumption in the whole head. Our novel technique may therefore aid in uncovering new properties of various drugs, which may impact metabolic rates. Furthermore, our method may give a better understanding of metabolic behavior in an experimental setup that more closely resembles physiological states.

Measuring alterations in metabolic rates is central to understanding the progression of various diseases and aging. Here, we present a novel technique to measure whole head oxygen consumption that more closely resembles the physiological state and may aid in revealing novel drugs that modify mitochondrial activity.

Regulated metabolic activity is essential for the normal functioning of living cells. Indeed, altered metabolic activity is causally linked with the ...

Analysis of Protein Folding, Transport, and Degradation in Living Cells by Radioactive Pulse Chase

Radioactive pulse-chase labeling is a powerful tool for studying the conformational maturation, the transport to their functional cellular location, and the degradation of target proteins in live cells. By using short (pulse) radiolabeling times (&lt;30 min) and tightly controlled chase times, it is possible to label only a small fraction of the total protein pool and follow its folding. When combined with nonreducing/reducing SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoprecipitation with (conformation-specific) antibodies, folding processes can be examined in great detail. This system has been used to analyze the folding of proteins with a huge variation in properties such as soluble proteins, single and multi-pass transmembrane proteins, heavily N- and O-glycosylated proteins, and proteins with and without extensive disulfide bonding. Pulse-chase methods are the basis of kinetic studies into a range of additional features, including co- and posttranslational modifications, oligomerization, and polymerization, essentially allowing the analysis of a protein from birth to death. Pulse-chase studies on protein folding are complementary with other biochemical and biophysical methods for studying proteins in vitro by providing increased temporal resolution and physiological information. The methods as described within this paper are adapted easily to study the folding of almost any protein that can be expressed in mammalian or insect-cell systems.

Here we describe a protocol for a general pulse-chase method that allows the kinetic analysis of folding, transport, and degradation of proteins to be followed in live cells.

Radioactive pulse-chase labeling is a powerful tool for studying the conformational maturation, the transport to their functional cellular location, and ...

Detection of Protein Ubiquitination Sites by Peptide Enrichment and Mass Spectrometry

The posttranslational modification of proteins by the small protein ubiquitin is involved in many cellular events. After tryptic digestion of ubiquitinated proteins, peptides with a diglycine remnant conjugated to the epsilon amino group of lysine (&#39;K-&#949;-diglycine&#39; or simply &#39;diGly&#39;) can be used to track back the original modification site. Efficient immunopurification of diGly peptides combined with sensitive detection by mass spectrometry has resulted in a huge increase in the number of ubiquitination sites identified up to date. We have made several improvements to this workflow, including offline high pH reverse-phase fractionation of peptides prior to the enrichment procedure, and the inclusion of more advanced peptide fragmentation settings in the ion routing multipole. Also, more efficient cleanup of the sample using a filter-based plug in order to retain the antibody beads results in a greater specificity for diGly peptides. These improvements result in the routine detection of more than 23,000 diGly peptides from human cervical cancer cells (HeLa) cell lysates upon proteasome inhibition in the cell. We show the efficacy of this strategy for in-depth analysis of the ubiquitinome profiles of several different cell types and of in vivo samples, such as brain tissue. This study presents an original addition to the toolbox for protein ubiquitination analysis to uncover the deep cellular ubiquitinome.

We present a method for the purification, detection, and identification of diGly peptides that originate from ubiquitinated proteins from complex biological samples. The presented method is reproducible, robust, and outperforms published methods with respect to the level of depth of the ubiquitinome analysis.

The posttranslational modification of proteins by the small protein ubiquitin is involved in many cellular events. After tryptic digestion of ...

Nitropeptide Profiling and Identification Illustrated by Angiotensin II

Protein nitration is one of the most important post-translational modifications (PTM) on tyrosine residues and it can be induced by chemical actions of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in eukaryotic cells. Precise identification of nitration sites on proteins is crucial for understanding the physiological and pathological processes related to protein nitration, such as inflammation, aging, and cancer. Since the nitrated proteins are of low abundance in cells even under induced conditions, no universal and efficient methods have been developed for the profiling and identification of protein nitration sites. Here we describe a protocol for nitropeptide enrichment by using a chemical reduction reaction and biotin labeling, followed by high resolution mass spectrometry. In our method, nitropeptide derivatives can be identified with high accuracy. Our method exhibits two advantages compared to the previously reported methods. First, dimethyl labeling is used to block the primary amine on nitropeptides, which can be used to generate quantitative results. Second, a disulfide bond containing NHS-biotin reagent is used for the enrichment, which can be further reduced and alkylated to enhance the detection signal on a mass spectrometer. This protocol has been successfully applied to the model peptide Angiotensin II in the current paper.

Proteomic profiling of tyrosine-nitrated proteins has been a challenging technique due to the low abundance of the 3-nitrotyrosine modification. Here we describe a novel approach for nitropeptide enrichment and profiling by using Angiotensin II as the model. This method can be extended for other in vitro or in vivo systems.

Protein nitration is one of the most important post-translational modifications (PTM) on tyrosine residues and it can be induced by chemical actions of ...