Table I. Exemplar turnover studies using stable isotope labels.

Experimental model	Stable isotope label	Labeling protocol	RIA transition	Calculation of turnover rate	Reference
Escherichia coli	[13C6]glucose	Labeled glucose added to cell culture. Cells harvested after 30 min.	Incorporation of label monitored. Single time point.	Synthesis/degradation ratio.	(68)
Mycoplasma pneumoniae	[13C6][15N4] arginine	Cells pre-labeled and transferred to unlabeled media for time-point sampling.	Loss of label monitored at 1, 2, 4, and 8 h.	TPP Xpress algorithm used to define H:L ratios over the time points. Protein half-lives determined.	(69)
Saccharomyces cerevisiae	dl-[2H10]leucine	Cells grown in glucose-limited culture media containing heavy leucine for seven doubling times. Media changed to unlabeled leucine for sampling.	Loss of label monitored at 0, 0.17, 0.67, 1, 2, 4, 6, 8, 10, 12, 25, and 51 h.	Non-linear curve fitting of (t, RIAt) data to obtain kloss. True rate of degradation (kdeg) by subtraction of the constant dilution rate D from kloss	(39)
Saccharomyces cerevisiae	([15N]H4)2SO4	Nitrogen-limited chemostat used to culture yeast. Inflowing media then changed to contain [15N] for 16 h.	Loss of unlabeled peaks monitored at 0, 2, 4.5, 7, 10, and 16 h.	Rate of protein degradation, assuming first-order kinetics, calculated by loss of unlabeled peptide peaks, and then half-life for each protein using t1/2 = ln2/kdeg	(40)
Corynebacterium glutamicum	[15N]H4Cl	Cells cultured in media with [15N]H4Cl as the sole nitrogen source until fully labeled and then transferred to unlabeled media and heat shocked.	Loss of label monitored at 0.5, 1, 2, and 4 h.	Calculation of protein synthesis rates were based on abundance ratios of new (partly [14N]-labeled) to old (fully [15N]-labeled) proteins.	(29)
Mycobacterium smegmatis	([15N]H4)2SO4	Cells grown in [15N] media until mid-log phase, washed, and then resuspended in [14N] high-iron medium or [14N] low-iron medium until OD doubled.	Loss of label monitored at 0, 2, 3, 4, 5, 6, and 7 h.	AL, AH, and AM represent the isotopologue intensities with light, heavy, and medium label. S/D = AM/AL or S/D = AL/AH.	(70)
Streptomyces coelicolor	[13C6][15N4] arginine	Cells prelabeled in heavy media and transferred to unlabeled medium.	Loss of label monitored at 0, 2, 4, and 8 h.	iTRAQ used to tag each of four time points for multiplexed turnover analysis.	(71)
Ostreococcus tauri	Na[15N]O3 and [15N]H4Cl	Cells grown in either labeled or unlabeled media, then resuspended in the opposite form of media.	Incorporation or loss of label monitored in the different cultures. Samples at 12 h, then 24 h intervals for 6 d.	Averaged peptide H/(H + L) values were plotted against time, enabling estimation of initial linear time-dependence by linear regression, where the slope (divided by 100) returns the initial turnover rate in %/h.	(72)
HeLa cells	l-[13C615N4]arginine, l-[13C615N2]lysine and l-[13C6]arginine, l-[2H4]lysine	Amino acids were added to cell cultures in light (Arg0, Lys0), medium (Arg6, Lys4), or heavy (Arg10, Lys8) form. Label incorporation assessed after six passages.	Labeling and unlabeling compared at 0.5, 4, 7, 11, 27, and 48 h.	Data analyzed using PepTracker turnover and spatial viewer. At each time point, labeled cells were mixed with those grown in light media.	(23)
Arrested HeLa or differentiated C2C12 cells	l-[13C615N4]arginine and l-[13C615N2]lysine	Cells initially grown in media with 1:1 Arg0/Lys0:Arg10/Lys8. Cells were arrested and then transferred to 100% heavy for 24 h.	At time t = 0, H = 50%. ΔH/(H + L) calculated so RIA values can be compared to standard RIA values where H = ΔH. Sampling at 1, 2, 4, 8, or 24 h.	Half-life t1/2 calculated using the loss of “light label” as a first-order process.	(26)
Human adenocarcinoma A549 cells	l-[13C6]arginine	Cells grown in label-containing media for 13 d, then resuspended and incubated in unlabeled media for chase.	Loss of label monitored at 0, 0.25, 0.5, 1, 2, 4, or 8 h.	First-order rate constants determined via nonlinear curve fitting.	(24)
HeLa cells	l-[13C615N4]arginine, l-[13C615N2]lysine and l-[13C6]arginine, l-[2H4]lysine	Light cells transferred to media containing heavy (H) or medium-heavy (M) arg/lys. Samples are combined for analysis.	Incorporation of labels monitored. Comparison of rates of protein translation between two samples by pulse labeling. Sampling at 1, 2, 4, 6, 8, and 10 h.	H vs. M peak intensity indicates translation differences between iron-treated cells.	(73)
Mouse fibroblasts	Heavy SILAC amino acids (not specified)	Cells grown initially in light amino acids and then pulse labeled with heavy amino acids.	Incorporation of label monitored at 1.5, 4.5, and 13.5 h.	Protein half-lives calculated from log H/L ratios at all three time points using linear regression.	(12)
Mouse (Mus musculus)	U-[13C6]glucose	Continuous venous infusion of glucose tracer over effective labeling phase of 8 h.	Single labeling time point. Liver proteins compared to control, unlabelled, liver.	Half-life (days) assessed as T1/2 = ln(2)/k/24	(66)
Mouse (Mus musculus)	[15N]-enriched Spirulina-based rodent diet	Animals acclimated to the unlabeled algae-based diet, then switched to labeled diet.	Incorporation of label monitored at nine times over 32 d in proteins from the brain, liver, and blood.	Turnover rate recovered from exponential fit post-labeling delay phase.	(43)
Mouse (Mus musculus)	[15N]-enriched Ralstonia eutropha protein-based rodent diet	Animals acclimated to the unlabeled diet, then diet changed to that containing labeled protein.	Incorporation of label monitored at 1, 2, 4, 7, and 14 d in brain and plasma proteins.	Direct analysis of free amino acids for enrichment of the precursor pool. Calculation of the labeled peptide fraction (LPF) using ProTurnyzer software. Delayed exponential curve: LPFt = 1 − e−λ(t − t0)	(44)
Mouse (Mus musculus)	[2H]2O	Two intraperitoneal injections of 99.9% saline [2H]2O, 4 h apart, then free access to 8% [2H]2O.	Incorporation of label monitored; heart, liver, and blood sampled at 0, 0.5, 1, 2, 4, 7, 12, 17, 22, 27, 32, 37, and 90 d for mitochondrial enrichment.	Non-linear curve fitting: A(t) = A(0) + A(∞) − A(0) (1 − e−kt), then transformed into fraction synthesis, f(t):(t) = ((t) − (0))/(A(∞) − A(0))=1 − e−kt to determine rate of turnover (k).	(25)
Mouse (Mus musculus)	[2H8]valine	Animals fed diet containing [2H8]valine at RIA of 0.5 after first acclimating to diet containing crystalline [H8]valine.	Incorporation of label monitored at 1, 2, 5, and 12 d. Liver, kidneys, heart, and skeletal muscle sampled from two animals per time point.	Precursor RIA calculated from divaline peptides via isotopomer analysis. (RIA, t) data analyzed by means of non-linear curve fitting.	(22)
Mouse (Mus musculus)	[2H8]valine	Animals fed diet containing [2H8]valine at RIA of 0.5 after first acclimating to diet containing crystalline [H8]valine.	Incorporation of label monitored at 2, 7, 14, 21, and 35 d. Samples of seminal vesicle secretions and sperm from two animals per time point.	Precursor RIA calculated from divaline peptides. First-order rate constant of labeling and the delay (days) in appearance of label caused by sperm maturation.	(74)
Mouse (Mus musculus)	[2H3] leucine	Animals fed unlabeled synthetic diet for 1 week, then transferred to deuterated leucine-containing diet.	Incorporation of label monitored at 3, 10, and 17 d for three mice per time point. Mitochondria from liver and heart analyzed.	Topograph software used to measure isotopologue abundances and calculate peptide turnover rates using precursor RIA and pt = p0 e−λ(t − t0)	(67)
Rat (Rattus norvegicus)	[15N]-enriched Spirulina algal cells	[15N]-algal cells fed to rats for two generations, starting either from before pregnancy or during pregnancy.	Level of incorporation in liver and brain proteins compared in pups from dams labeled either pre- vs. during pregnancy.	Incorporation levels compared for different proteins.	(63)
Rat (Rattus norvegicus)	[2H]2O	Intraperitoneal injection of [2H]2O to enrich body water to 2.6% and drinking water replaced with 5% [2H]2O. Two groups of animals, fed and fasted.	Incorporation of label monitored; blood and plasma samples collected after 30 and 60 min, then at 1 h intervals for 8 h.	Fractional synthesis rate determined using a one-compartment model. Time course of total labeling of a peptide (Epeptide(t)) fit to exponential rise curve equation: Epeptide(t) = Ess * (1 − e−kt)	(75)
Rat (Rattus norvegicus)	[15N]-enriched algal cells	Female rats and offspring fed [15N] diet for 6 weeks. Offspring changed to [14N] diet after this time.	Dam rats culled when fully labeled. Proteins from liver and brain of progeny rats analyzed after 6 and 12 months.	[15N]/[14N] calculated at sampling times.	(31)
Zebrafish (Danio rerio)	[13C6]lysine	Fish were fed 2:1 ratio of [13C6]lysine:normal fish feed for 4 months. Fish split into two groups and then fed unlabeled or [13C6]lysine containing diet.	Labeling and unlabeling of brain and re-grown amputated fins compared.	SILAC-type analysis.	(45)
Common carp (Cyprinus carpio)	l-[2H7]leucine	Synthetic diet containing unlabeled leucine fed to fish twice per day for 4 weeks. Diet then switched to contain deuterated leucine for up to 7 weeks.	Incorporation of label into muscle proteins monitored at 0, 1, 2, 3, 4, 5, and 7 weeks.	Precursor pool RIA calculated from parvalbumin peptides to allow synthesis and degradation rates to be determined through non-linear curve fitting.	(76)
Chicken (Gallus gallus)	[2H8]valine	Animals fed diet containing [2H8]valine at RIA of 0.5 for 120 h.	Incorporation of label into skeletal muscle proteins monitored at 0, 1, 2, 4, 8, 14, 24, 30, 32, 48, 55, 72, 96, and 120 h for three chickens at each time point.	Precursor RIA from multiply labeled peptides applied to non-linear curve fitting. Data adjusted to correct for muscle growth.	(42)
Mouse (Mus musculus), rat (Rattus norvegicus), and human (Homo sapiens) studies	[2H]2O	Bolus injection (intraperitoneal) of [2H]2O followed by [2H]2O drinking water for all studies. Samples for body water enrichment taken at various times. In rat “decay of 2H” study, animals labeled for 8 to 10 weeks; then water changed to unlabeled.	Loss and incorporation of label monitored in variety of different samples (bone, muscle, liver, sera) and species.	MIDA calculations and GC/MS to determine RIA of M0, M1, and M2 isotopomers. Fractional protein synthesis rates, or label decay rates estimated by non-linear least squares fitting. Half-lives calculated as t = 0.693/k.	(35)
Human (Homo sapiens)	[2H]2O	Short-term (7 day) study: oral dosing of eight 50 ml 70% [2H]2O over 36 h, then two doses every 24 h.	Incorporation of label monitored via daily blood sampling.	Fractional synthesis (proportion of new protein as a fraction of total pool) and rate constant for protein turnover using SAAM program to calculate time-dependant changes in isotopic enrichment of newly synthesized peptides.	(36)
		Long-term (6 weeks) study: oral administration of three doses per day for 7 days, then two per day.