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.
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Comparison of dynamic SILAC and TMT-SILAC hyperplexing for measurement of protein turnover.
A, In dynamic SILAC experiments, the fractional labeling of proteins is monitored at the MS1 level. During the course of isotopic labeling, unlabeled proteins ( green) are cleared and replaced by labeled proteins ( orange). Fractional labeling is measured by analyzing the spectra of intact peptide ions over time in distinct LC-MS/MS runs. Resulting curves for either the decay of unlabeled peptides or appearance of labeled peptides are fitted to exponential equations to obtain the turnover rate constants or half-lives of proteins. B, In TMT-SILAC hyperplexing, experimental samples from different SILAC labeling time-points are reacted with different TMT tags and combined in a single multiplexed sample prior to LC-MS/MS. At different time-points, a peptide generated from the same protein will have identical (overlapped) masses in MS1 spectra but will generate reporter ions with distinct masses in MS2 spectra. Thus, fragmentation of SILAC-unlabeled peaks will provide the kinetics of decay for unlabeled peptides and fragmentation of SILAC-labeled peaks will provide the kinetics of appearance for labeled peptides.
TMT-SILAC hyperplexing experimental design and sample data.
A, Experimental design. HCA2-hTert cells were grown to confluency and SILAC labeling was conducted over the course of 336 h as cells were in a contact-inhibited quiescent state. Extracts were collected from 10 time-points and, following trypsinization, each was labeled with a different TMT tag. The TMT-tagged peptides were combined in a single multiplexed sample and analyzed by LC-MS/MS as described under Experimental Procedures. Additionally, a number of TMT-tagged time-points ( boxed) were individually analyzed by LC-MS/MS without multiplexing using a standard dynamic SILAC approach. B, The fractional labeling of a tryptic peptide ion (GEYDVTVPK, z = 2) belonging to neuroblast differentiation-associated protein analyzed at the MS1 level by dynamic SILAC. The green and orange peaks indicate the relative intensities of unlabeled and labeled peptides, respectively. The plot indicates the decay curve for the fractional labeling of the unlabeled peaks for GEYDVTVPK ( green circles) and all other peptides mapped to the protein ( transparent blue circles). The green line indicates a fit to a single exponential equation. The fractional labeling of labeled peaks (calculated as one minus the fractional labeling of unlabeled peaks) is indicted by a dotted orange line. C, The kinetics for clearance of GEYDVTVPK analyzed at the MS2 level by TMT-SILAC hyperplexing. The MS2 spectrum indicates the relative intensities of TMT reporter ions for the SILAC-unlabeled peptide ion. The plot indicates intensities of peaks relative to the t0 sample (TMT-126) for GEYDVTVPK ( green circles) and all other peptides mapped to the protein ( transparent blue circles). The green line indicates a fit to a single exponential equation with a non-zero baseline indicated by a dashed line and double-headed arrow. D, The kinetics for appearance of GEYDVTVPK analyzed at the MS2 level by TMT-SILAC hyperplexing. The MS2 spectrum indicates the relative intensities of TMT reporter ions for the SILAC-labeled peptide ion. The plot indicates intensities of peaks relative to the fitted intensity of the exponential curve ( orange line) at infinite time ( t ∞). The dashed line indicates the intensity of the t0 sample (TMT-126) and represents the initial baseline of the curve. E and F, Data analyzed as in C and D, respectively, except at the MS3 level using SPS-MS3.
Labeling of different peptides mapped to the same protein have similar kinetics. As examples, the traces in the plot ( A) indicate exponential fits to peptide data for two different proteins with varying degradation rates, ACTB and AHNAK. The data was collected from MS2 TMT reporter tags of SILAC labeled precursors. The boxplots ( B) provide a global analysis of variance in rate measurements between peptides belonging to the same protein. The box shows the median and interquartile range (IQR) of the coefficient of variations (CV) of degradation rates for peptides encompassing a single protein for all methods. The error bar represents the entire range of CVs excluding outliers (>1.5 IQR). The white lines and numbers represent mean values. The red circles indicate the CV among all measured peptide degradation rates for each method. The analysis was limited to proteins shared between all analyses.
Pairwise comparison of The analysis was limited to proteins shared between all analyses. Spearman rank correlation coefficients are indicated. The identity line is shown in red. k measurements obtained by TMT-SILAC hyperplexing and dynamic SILAC. deg
The use of TMT-SILAC hyperplexing to measure relative differences in steady-state protein levels, degradation rates (
k) and synthesis rates ( deg k) between proliferating and quiescent cells. syn A, The schematic illustrates the kinetic model for synthesis and clearance of proteins in quiescent and proliferating cells. B, A theoretical TMT-SILAC hyperplexing analysis of a hypothetical protein with varying k and syn k values in quiescent and proliferating cells. The analysis of the decay of SILAC-unlabeled peaks allows the measurement of deg k for both conditions as well as the ratio of steady-state protein levels. These parameters ( deg boxed in the equation at the bottom of the figure) enable the measurement of the ratio of k between the two conditions. The equation is derived based on the model presented under Experimental Procedures. syn C, Experimental analysis of an example protein (Glyceraldehude-3-Phosphate) showing the measured values for ratios of k, syn k and steady-state protein levels. The deg plot indicates the decay curve for the fractional labeling of SILAC-unlabeled peaks for all peptides mapped to the protein ( transparent circles) as well as the median value for all peptides ( solid circles). The solid line indicates a fit to a single exponential equation for measurement of clearance rate constants. For quiescent cells, the clearance rate constant equals the degradation rate constant. For proliferating cells, the rate of cell division (0.40 d −1) is subtracted from the clearance rate in order to determine the degradation rate ( dashed blue line). D, The distribution of log2 ratios of synthesis and degradation rate constants between quiescent and dividing cells. The histograms are shown for all proteins, long-lived proteins (half-lives greater than 3 days in proliferating cells) and short-lived proteins (half-lives less than 3 days in proliferating cells). The dotted lines on the rightmost plot indicate the distribution of long-lived proteins for comparison.
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No abstract available.
Cell Culture Techniques / methods*
Isotope Labeling / methods*