SILAM Mouse Tissues

Relative quantification of the mouse proteome and metabolome using the SILAM spike-in approach

The central element of the SILAM method is the “spike-in” approach: Differences in protein patterns of unlabeled case tissue with respect to unlabeled control tissue can be quantified by “spiking-in” a stable-isotope-labeled reference tissue.This stable-isotope labeled reference tissue can either be cultivated using the appropriate feed (see SILAM diets), or it can be obtained directly from Silantes.

Silantes offers various unlabeled and stable isotope-labeled mouse tissues as a reference for the SILAM spike-in approach:

¹³C lysine labeled SILAM mouse tissues (suitable for proteomics studies)
¹⁵N labeled SILAM mouse tissues (suitable for proteomics and metabolomics studies)

Overview

The SILAM “spike-in” workflow
New: Lyophilized Reference Tissues from Silantes
Advantages of using lyophilized tissue as a reference for the SILAM method
References

The SILAM “spike-in” workflow

A short outline of the SILAM “spike-in” procedure

Figures 1 and 2 show the SILAM spike-in workflow for different labeling strategies: Differences in protein levels of unlabeled tissue (A) with respect to unlabeled tissue (B) can be quantified by “spiking-in” an isotopically-labeled reference sample (R). The reference tissue can be obtained from Silantes, as frozen or lyophilized material.

Figure 1: ¹³C-Lysine labeled SILAM tissue spike-in workflow

Figure 2: ¹⁵N labeled SILAM tissue spike-in workflow

The isotopically labeled “heavy” reference tissue protein or metabolite extract (R) is mixed with the unlabeled “light” tissue extract (A) and (B), respectively. The proteomes or metabolomes of the (A)(R)-mix and (B)(R)-mix are isolated, processed and subjected to LC-MS which enables the relative quantification of (A/B).

Calculating the ratio (A/R) : (B/R) cancels out the reference amount (R) and yields the ratio of peptides or metabolites (A/B). The stable isotopically labeled mouse tissue (R) is used as a standard, permitting normalizing (A) with respect to (B), accounting for differences in the isolation procedure in the mixtures (A/R) and (B/R) without affecting the peptide or metabolite ratios (A/B). Therefore, the mouse strain (R) must not necessarily be the same mouse strain as (A) and (B).

Conclusion:
The customer can use any mouse strain in combination with the Silantes stable isotope-labeled mouse tissue as a reference.

New: Lyophilized Reference Tissues from Silantes

Silantes has recently developed a cost-efficient alternative to the stable isotope-labeled wet reference tissues in the form of lyophilized reference tissues. This improves the handling of the reference and is more convenient for quantifying protein patterns of single mouse organs.

The lyophilized reference tissue is prepared as a ready-to-use sample: The isotope-labeled proteins of the corresponding mouse organ are already extracted (optionally with SDS or urea). The sample contains 100 µg of protein which is sufficient for about 3 experiments.

Advantages of using lyophilized tissue as a reference for the SILAM method

Improved handling with unchanged high quality

Silantes conducted a comparative study of wet and lyophilized references in cooperation with Prof. Marcus Krüger from Max Planck Institute for Heart and Lung Research. The results show that the wet sample can be replaced by the lyophilized sample without loss of accuracy. Figure 3 shows the analysis of the SDS gel patterns of wet liver (STD) and lyophilized liver (Lyo).

Figure 3: SDS-PAGE of liver tissue samples: Standard (wet liver) and Lyo. (lyophilized liver)

To verify that lyophilization does not change the protein content with respect to the wet sample, both samples were analyzed by mass spectrometry and their peptide patterns were compared in figure 4:

Row (1) shows the ratio of ¹³C-labeled (H) to unlabeled (L) peptides of the wet sample (STD); Row (2) shows the same for the lyophilized tissue (Lyo.) and row (3) shows the direct comparison of “STD.” and “Lyo.”. A comparison of the data in row (1) and row (2) shows that the peptide distributions of “STD” and “Lyo.” have a high degree of similarity. An even greater similarity is observed if wet tissue and lyophilized tissue are directly compared in row (3).

Figure 4: Ratio of labeled (H) to unlabeled (L) peptides of the STD and Lyo.

Figure 5 shows a correlation plot using the same data as in Figure 4. The pearson correlation coefficient of 0.83 indicates a high level of correlation between the peptide data sets from the wet tissue (STD) and the lyophilized tissue (Lyo.), supporting the view that lyophilized tissue is a suitable alternative to the wet tissue.

Figure 5: Correlation plot of labeled (H) to unlabeled (L) peptides of the STD and Lyo.

Figure 6 shows the correlation with the same samples as above, namely the wet liver (STD) and the lyophilized liver (Lyo.), including a new unrelated liver sample (#2) in wet and lyophilized form. As expected, the data show a lower correlation of #1 with respect to #2 indicating that differences between unrelated liver samples can be detected.

Figure 6: Correlation plot including a new liver sample in wet and lyophilized form

References:

Relevant documents:

Use cases of the SILAM amino acid mouse diet Silantes in scientific publications:

Silantes Lys6-SILAM diet (Complete labeling of oocytes): Harasimov, K., Gorry, R. L., Welp, L. M., Penir, S. M., Horokhovskyi, Y., Cheng, S., Takaoka, K., Stützer, A., Frombach, A., Tavares, A. L. T., Raabe, M., Haag, S., Saha, D., Grewe, K., Schipper, V., Rizzoli, S. O., Urlaub, H., Liepe, J., & Schuh, M. (2024). The maintenance of oocytes in the mammalian ovary involves extreme protein longevity. Nature Cell Biology, 26(7), 1124–1138. https://doi.org/10.1038/s41556-024-01442-7
Silantes Lys6-SILAM diet (Time dependent labeling of sceletal mucle tissue): Bock, T., Türk, C., Aravamudhan, S. et al. PERM1 interacts with the MICOS-MIB complex to connect the mitochondria and sarcolemma via ankyrin B. Nat Commun 12, 4900 (2021). https://doi.org/10.1038/s41467-021-25185-3
Silantes Lys8-SILAM diet (Time dependent labeling of liver, skeletal muscle, cartilage, mucosa, and blood): Rolfs, Z., Frey, B.L., Shi, X. et al. An atlas of protein turnover rates in mouse tissues. Nat Commun 12, 6778 (2021). https://doi.org/10.1038/s41467-021-26842-3
Silantes Lys6-SILAM diet (Time dependent labeling of spinal cord tissue): Meschkat, M., Steyer, A.M., Weil, MT. et al. White matter integrity in mice requires continuous myelin synthesis at the inner tongue. Nat Commun 13, 1163 (2022). https://doi.org/10.1038/s41467-022-28720-y
Silantes Lys6-SILAM diet (Complete labeling of heart tissue): Aravamudhan, S., Türk, C., Bock, T., Keufgens, L., Nolte, H., Lang, F., Krishnan, R. K., König, T., Hammerschmidt, P., Schindler, N., Brodesser, S., Rozsivalova, D. H., Rugarli, E., Trifunovic, A., Brüning, J., Langer, T., Braun, T., & Krüger, M. (2021). Phosphoproteomics of the developing heart identifies perm1 – an outer mitochondrial membrane protein. Journal of Molecular and Cellular Cardiology, 154, 41–59. https://doi.org/10.1016/j.yjmcc.2021.01.010
Silantes Lys6-SILAM diet (Time dependent labeling of kidney tissue): Rinschen, M. M., Gödel, M., Grahammer, F., Zschiedrich, S., Helmstädter, M., Kretz, O., Zarei, M., Braun, D. A., Dittrich, S., Pahmeyer, C., Schroder, P., Teetzen, C., Gee, H., Daouk, G., Pohl, M., Kuhn, E., Schermer, B., Küttner, V., Boerries, M., Huber, T. B. (2018). A multi-layered quantitative in vivo expression atlas of the podocyte unravels kidney disease candidate genes. Cell Reports, 23(8), 2495–2508. https://doi.org/10.1016/j.celrep.2018.04.059
Silantes Lys6-SILAM diet (Time dependent labling of kidney tissue): Rinschen, M. M., Palygin, O., El-Meanawy, A., Domingo-Almenara, X., Palermo, A., Dissanayake, L. V., Golosova, D., Schafroth, M. A., Guijas, C., Demir, F., Jaegers, J., Gliozzi, M. L., Xue, J., Hoehne, M., Benzing, T., Kok, B. P., Saez, E., Bleich, M., Himmerkus, N., Staruschenko, A. (2022). Accelerated lysine metabolism conveys kidney protection in salt-sensitive hypertension. Nature Communications, 13(1). https://doi.org/10.1038/s41467-022-31670-0
Silantes Lys6-SILAM diet (Time dependent labeling of epithelial cells and mucus along the gastrointestinal tract): Arike, L., Seiman, A., van der Post, S., Rodriguez Piñeiro, A. M., Ermund, A., Schütte, A., Bäckhed, F., Johansson, M. E. V., Hansson, G. C. (2020). Protein turnover in epithelial cells and mucus along the gastrointestinal tract is coordinated by the spatial location and microbiota. Cell Reports, 30(4). https://doi.org/10.1016/j.celrep.2019.12.068
Silantes Lys6-SILAM diet (Time dependent labeling of brain tissue): Andrews, B., Murphy, A. E., Stofella, M., Maslen, S., Almeida-Souza, L., Skehel, J. M., Skene, N. G., Sobott, F., Frank, R. A. W. (2022). Multidimensional Dynamics of the proteome in the neurodegenerative and aging mammalian brain. Molecular and Cellular Proteomics, 21(2), 100192. https://doi.org/10.1016/j.mcpro.2021.100192
Silantes Lys6-SILAM diet (Time dependent labeling of muscle tissue): Kallabis S, Abraham L, Müller S, Dzialas V, Türk C, Wiederstein JL, Bock T, Nolte H, Nogara L, Blaauw B, Braun T, Krüger M. High-throughput proteomics fiber typing (ProFiT) for comprehensive characterization of single skeletal muscle fibers. Skelet Muscle. 2020 Mar 23;10(1):7. doi: 10.1186/s13395-020-00226-5. PMID: 32293536; PMCID: PMC7087369

Use cases of the SILAM 13C and 15N mouse diet from Silantes in scientific publications:

Silantes 15N SILAM diet (Imaging using NanoSims, time dependent proteomics of retina tissue): Bonnin, E. A., Fornasiero, E. F., Lange, F., Turck, C. W., & Rizzoli, S. O. (2021). NanoSIMS observations of mouse retinal cells reveal strict metabolic controls on nitrogen turnover. BMC Molecular and Cell Biology, 22(1). https://doi.org/10.1186/s12860-020-00339-1
Silantes 15N SILAM diet (time dependent targeted metabolomics and proteomics): Weckmann, K., Deery, M. J., Howard, J. A., Feret, R., Asara, J. M., Dethloff, F., Filiou, M. D., Labermaier, C., Maccarrone, G., Lilley, K. S., Mueller, M., & Turck, C. W. (2018). Ketamine’s effects on the glutamatergic and GABAergic systems: A proteomics and metabolomics study in mice. Complex Psychiatry, 5(1), 42–51. https://doi.org/10.1159/000493425
Silantes 15N-SILAM diet (time dependent targeted metabolomics and proteomics, brain tissue, hippocampus tissue, blood tissue, plasma): Zhang, Y., Filiou, M. D., Reckow, S., Gormanns, P., Maccarrone, G., Kessler, M. S., Frank, E., Hambsch, B., Holsboer, F., Landgraf, R., & Turck, C. W. (2011). Proteomic and metabolomic profiling of a trait anxiety mouse model implicate affected pathways. Molecular & Cellular Proteomics, 10(12). https://doi.org/10.1074/mcp.m111.008110
Silantes 15N-SILAM diet (complete labeling of brain tissue, proteomics): Klingener, Michael, et al. “N-Cadherin Promotes Recruitment and Migration of Neural Progenitor Cells from the SVZ Neural Stem Cell Niche into Demyelinated Lesions.” The Journal of Neuroscience, vol. 34, no. 29, 16 July 2014, pp. 9590–9606, https://doi.org/10.1523/jneurosci.3699-13.2014.
Silantes 15N-SILAM diet (time dependent labeling in slowly proliferative tissues, proteomics): John Hasper, Kevin Welle, Jennifer Hryhorenko, Sina Ghaemmaghami, Abigail Buchwalter Turnover and replication analysis by isotope labeling (TRAIL) reveals the influence of tissue context on protein and organelle lifetimes. Mol Syst Biol. (2023) 19: e11393 https://doi.org/10.15252/msb.202211393

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