SILAC 代谢标记系统

Thermo Fisher Scientific offers the broadest portfolio of liquid and powdered SILAC media and SILAC amino acids. The media and amino acids are available in kits, or as stand-alone reagents, for labeling proteins expressed in a wide variety of mammalian cell lines, including HeLa, 293T, COS7, U2OS, A549, NIH 3T3, Jurkat, and others.
 

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The SILAC Protein Quantitation kits enable successful isotope metabolic protein labeling for relative quantitation of protein abundance in different cell treatments, and expression profiling of normal vs. disease cells. Two types of kits are available that have been optimized for use with LysC or trypsin for protein digestion.

• LysC SILAC Kits are supplied with "heavy" L-Lysine (13C6 L-Lysine-2HCl) to label and quantify lysine-containing peptides from LysC protein digests. LysC SILAC kits can be converted to a trypsin SILAC kit by substituting "light" L-arginine, with "heavy" L-arginine, 13C6 L-Arginine-HCl or 13C6 15N4 L-Arginine-HCl.
• Trypsin SILAC kits are supplied with "heavy" L-Lysine (13C6 15N2 L-Lysine-2HCl) and "heavy" L-arginine (13C6 15N4 L-Arginine-HCl) to label and quantify lysine-containing and arginine-containing peptides from trypsin protein digests.

Compared to "light" peptides, +2 ionized, heavy isotope peptides containing 13C6 L-Lysine or 13C6 15N4 L-Arginine get shifted by 3 and 5 m/z (Figure 1).

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Normalized protein extracts isolated from cells are combined, reduced, alkylated, and digested overnight. For the in-gel workflow, samples are run on an SDS-PAGE gel, excised, digested, and cleaned up while for the in-solution workflow, samples are digested, fractionated, and cleaned up. Samples are then analyzed by high-resolution Orbitrap LC-MS/MS (Figure 2).

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The heavy and light amino acids are used to specifically analyze protein expression by mass spectrometry using stable isotope labeling with amino acids in cell culture (SILAC) quantification kits.

General features of heavy and light amino acids for SILAC labeling:

• Efficient—100% label incorporation into proteins of living cells
• Flexible—different isotopes of heavy and/or light amino acids for arginine, lysine, leucine, and proline enable the quantitation of peptides derived from MS-grade proteases
• Multiplex capabilities—several alternative isotopes of arginine and lysine are available that allow the analysis of multiple treatment conditions in each experiment
• High-quality isotope enrichment—heavy amino acids with > 99% isotope purity

Heavy and light amino acids for SILAC are used together with specialized cell culture media that are deficient in essential amino acids. Heavy and light L-lysine and L-arginine are the most common amino acids used for SILAC analysis of tryptic peptides. Up to 3 different experimental conditions can be readily analyzed with different isotopes of lysine and arginine. For lysine 3-plex experiments, 4,4,5,5-D4 L-lysine and 13C6 15N2 L-lysine are used to generate peptides with 4 Da and 8 Da mass shifts, respectively, compared to peptides generated with light lysine. For arginine 3plex experiments, 13C6 L-arginine and 13C6 15N4 L-arginine are used to generate peptides with 6 Da and 10 Da mass shifts, respectively, compared to peptides generated with light arginine. L-leucine, a common amino acid found in protein sequences is also used for SILAC labeling. Proline, a non-essential amino acid is sometimes added to SILAC media to prevent the metabolic conversion of heavy arginine to heavy proline in mammalian cell lines with high arginine dehydrogenase activity.

Table 1. List of SILAC isotopes of amino acids for multiplex experiments

*Deuterium

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Cellular protein turnover is the net result of protein synthesis and degradation, and is crucial to maintain protein homeostasis and cellular function under steady-state conditions and to enable cells to remodel their proteomes upon a perturbation.

The kinetics and dynamics of protein turnover helps us understand how cells regulate essential processes such as growth, differentiation, and stress response. The quantitative approach to study proteome turnover is called dynamic SILAC which involves only two SILAC channels, "light" and "heavy". Briefly, cells are switched from unlabeled medium to a medium containing isotopically labeled amino acids, still typically heavy lysine and/or arginine [1]. Samples are then measured via liquid chromatography coupled to tandem mass spectrometry (LCMS/MS) over a time course. The ratio of heavy to light peptide signal thus directly reflects protein turnover (Figure 3) [2].

Ideally, quantitative analyses of proteome turnover and protein half-lives include data on both protein synthesis and degradation rates separately. The second strategy to measure protein production and degradation separately and track their changes upon perturbation is to combine dynamic SILAC with isobaric labeling (dynamic SILAC-TMT, Figure 3) [3,4,5,6]. Combining isobaric labels and dynamic SILAC facilitates direct quantification of heavy and light isotope–derived peaks, and also enables multiplexed analyses [3,4,5,6].

Dynamic SILAC has been performed in human A549 adenocarcinoma cells [1], rat neurons, and glia [7]. In vivo proteome turnover rates have been elucidated by feeding isotopically-labeled amino acids to whole animals such as C. elegans [8, 9], Drosophila [10], Zebrafish [11,12] and mice [13,14,15].

Figure 3. The dynamic SILAC workflow. (A) Sample prep for a standard two-label dynamic SILAC experiment. Cultures are plated in unlabeled media, which is swapped with media containing stable isotope–labeled amino acids (e.g., 13C6 ,15N4-Arg “heavy” arginine). Samples are collected and harvested over a time course. After sample digestion and purification, isobaric TMT labeling can be done to multiplex samples from different conditions and time points of interest. A fully labeled sample using a third stable isotope, typically a "semi-heavy /medium-heavy" isotope (e.g., 13C6-Arg), can also be generated as normalization standard for data analysis. (B) Data acquisition by LC-MS/MS. Direct monitoring of "light" (red) and "heavy" (green) peptide signals, corresponding to pre-existing and newly synthesized proteins is shown. In dynamic SILAC-TMT experiments, relative quantification of each sample is completed at the MS2 level (far right). In 3-channel designs, the signal from the constant "semi-heavy"-labeled sample (yellow) spike-in provides an internal normalization standard between different mass spectrometry measurements, allowing for relative signal from "light" and "heavy" channels to be quantitated. (C) Data analysis. Protein was measured from a 2-channel dynamic SILAC (left), a 3-channel dynamic SILAC (middle), and a combined 2-channel dynamic SILAC-TMT experiment (right). In the case of the latter, all the "heavy" (H) and "light" (L) signals are measured in the same run, which allows for separate synthesis and degradation curves.

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1. Doherty M.K., Hammond D.E., Clague M.J., et al. Turnover of the human proteome: determination of protein intracellular stability by dynamic SILAC. J Proteome Res, 2009. 8(1):104–112. 
2. Ross A.B., Langer J.D., Jovanovic M. Proteome Turnover in the Spotlight: Approaches, Applications, and Perspectives. Mol Cell Proteomics, 2021. 20:100016. 
3. Welle K.A., Zhang T., Hryhorenko J.R., et al. Time-resolved Analysis of Proteome Dynamics by Tandem Mass Tags and Stable Isotope Labeling in Cell Culture (TMT-SILAC) Hyperplexing. Mol Cell Proteomics, 2016. 15(12):3551–3563. 
4. Zecha J., Meng C., Zolg D.P., et al. Peptide Level Turnover Measurements Enable the Study of Proteoform Dynamics. Mol Cell Proteomics, 2018. 17(5):974–992. 
5. Savitski M.M., Zinn N., Faelth-Savitski M., et al. Multiplexed Proteome Dynamics Profiling Reveals Mechanisms Controlling Protein Homeostasis. Cell, 2018. 173(1):260–274. 
6. Jayapal K.P., Sui S., Philp R.J., et al. Multitagging proteomic strategy to estimate protein turnover rates in dynamic systems. J Proteome Res, 2010. 9(5):2087–2097. 
7. 7. Dörrbaum A.R., Schuman E.M., Langer J.D. Dynamic SILAC to Determine Protein Turnover in Neurons and Glia. Methods Mol Biol, 2023. 2603:1–17. 
8. Visscher M., De Henau S., Wildschut M.H.E., et al. Proteome-wide Changes in Protein Turnover Rates in C. elegans Models of Longevity and Age-Related Disease. Cell Rep, 2016. 16(11):3041–3051. 
9. Dhondt I., Petyuk V.A., Bauer S., et al. Changes of Protein Turnover in Aging Caenorhabditis elegans. Mol Cell Proteomics, 2017. 16(9):1621–1633. 
10. Rosenberger F.A., Atanassov I., Moore D., et al. Stable Isotope Labeling of Amino Acids in Flies (SILAF) Reveals Differential Phosphorylation of Mitochondrial Proteins Upon Loss of OXPHOS Subunits. Mol Cell Proteomics, 2021. 20:100065.
11. Westman-Brinkmalm A., Abramsson A., Pannee J., et al. SILAC zebrafish for quantitative analysis of protein turnover and tissue regeneration. J. Proteomics, 2011. 75(2), 425–434. 
12. Nolte H., Hölper S., Housley M.P. et al. Dynamics of zebrafish fin regeneration using a pulsed SILAC approach. Proteomics, 2015. 15(4), 739–751. 
13. Mandad S., Rahman R.U., Centeno T.P., et al. The codon sequences predict protein lifetimes and other parameters of the protein life cycle in the mouse brain. Sci Rep, 2018. 8(1), 16913. 
14. Fornasiero E.F., Mandad S., Wildhagen H., et al. Precisely measured protein lifetimes in the mouse brain reveal differences across tissues and subcellular fractions. Nat Commun, 2018. 9(1), 4230. 
15. Alevra M., Mandad, S., Ischebeck, T., et al. A mass spectrometry workflow for measuring protein turnover rates in vivo. Nat. Protoc, 2019. 14(12), 3333–3365. 

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