SILAM
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Stable Isotope Labeling in Mammals (SILAM)
John R. Yates, III, PhD
Ernest W. Hahn Professor
The Scripps Research Institute, Chemical Physiology & Cell Biology
SILAM refers to labeling an entire rodent with heavy stable isotopes for quantitative proteomic tissue analysis. Labeling an entire proteome with heavy isotopes in vivo generates an ideal standard for quantitative proteomics. When a heavy labeled proteome is mixed with an unlabeled proteome then digested, every unlabeled peptide identified by the mass spectrometer can be quantified by its corresponding heavy peptide. The advantage of metabolic labeling over in vitro tagging techniques is that the heavy and unlabeled samples are mixed before sample preparation, preventing variability between preparations from distorting the quantification results. This is especially important when extensive sample preparation (e.g. isolation of an organelle) is required. In SILAM, the rodent food is altered to contain heavy lysine or 15N spirulina as the only protein source. The heavy tissues are used as internal standards for quantitative proteomic analysis of basic mammalian physiology and animal models of disease.
Resources
Mouse Express NeuCode™ Mouse Feed
For more information on NeuCode™ and its applications, please visit Dr. Josh Coon's NeuCode-focused webpage at National Center for Quantitative Biology of Complex Systems.
Frequently Asked Questions
What is contained in the Mouse Express® Mouse Feed Kits?
The mouse feed kits contain 1 kg of labeled feed (e.g., 13C6, L-lysine, D3 L-leucine, 15N spirulina) and 1 kg of unlabeled feed. Please inquire if alternate quantities or formulations are required for your research studies.
Can irradiated Mouse Express® mouse feed be provided?
Yes. In this process, the feed is exposed to gamma radiation (cobalt-60 at 20 to 50 kGy) to reduce the bioburden and provide enhanced microbial control. Note that the irradiated feed contains an additional supplement of vitamins and amino acids. This helps compensate for their destruction that may occur during irradiation. Also note that irradiated feeds take 4-5 weeks to produce, while nonirradiated feeds take 2-3 weeks.
Will autoclave or gamma irradiation impact the mouse feed composition?
These diets will not withstand autoclaving, both from a physical and nutritional standpoint. Irradiation (at 20-50 kGy dose) impacts the composition; however, the loss is compensated for by an increase in certain components (e.g., vitamins).
What is the macronutrient information for a representative spirulina 15N-containing diet?
What is the macronutrient information for an amino acid-defined diet lysine?
What quality control measures are performed on spirulina?
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• Appearance |
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• GC/MS (for identification) |
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• GC/MS (for isotopic enrichment) |
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• ICP analysis (for the presence of metals) |
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• Nitrates / nitrites |
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• Moisture, protein, fat, fiber, ash, mineral content |
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• Protein based on amino acid content |
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• Protein from Kjeldahl nitrogen (KN) |
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• pH analysis |
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• Brine shrimp toxicity test |
What are the shipping and storage conditions of the mouse feeds?
The feeds are shipped in vacuum sealed pouches (in 1 kg units) at ambient temperature. It is recommended that these be stored at 4°C. It should be noted that the diets may be frozen; however, transferring a diet to refrigeration prior to room temperature is recommended in order to reduce the formation of condensation.
What are the merits of 15N labeling in SILAM?
Metabolic incorporation of 15N enables broad 'omic analysis (e.g., proteomic, metabolic, and glycomic) and a wide selection of 15N-labeled peptides (with 15N labeling on backbone and side chains) for quantitative MS analysis.
References
Ma, Y.; McClatchy, D.B.; Martínez-Bartolomé, S.; et al. 2021. Temporal quantitative profiling of newly synthesized proteins during Aβ accumulation. J Proteome Res, 20(1), 763-775. PMID: 33147027
Hark, T.J.; Rao, N.R.; Castillon, C.; et al. 2020. Pulse-chase proteomics of the APP knockin mouse models of Alzheimer's disease reveals that synaptic dysfunction originates in presynaptic terminals. Cell Syst, S2405-4712(20), 30458-30460. PMID: 33326751
Jongkamonwiwat, N.; Ramirez, M.A.; Edassery, S.; et al. 2020. Noise exposures causing hearing loss generate proteotoxic stress and activate the proteostasis network. Cell Rep, 33(8), 108431. PMID: 33238128
Liu, P.; Xie, X.; Jin, J. 2020. Isotopic nitrogen-15 labeling of mice identified long-lived proteins of the renal basement membranes. Sci Rep, 10(1), 5317. PMID: 32210336
Drigo, R.A.E.; Lev-Ram, V.; Tyagi, S.; et al. 2019. Age mosaicism across multiple scales in adult tissues. Cell Metab, 30(2), 343-351. PMID: 31178361
Wallace, M.; Green, C.R.; Roberts, L.S.; et al. 2018. Enzyme promiscuity drives branched-chain fatty acid synthesis in adipose tissues. Nat Chem Biol, 14(11), 1021-1031. PMID: 30327559
Heo, S.; Diering, G.H.; Na, C.H.; et al. 2018. Identification of long-lived synaptic proteins by proteomic analysis of synaptosome protein turnover. Proc Natl Acad Sci U S A, 115(16), E3827-E3836. PMID: 29610302
Overmyer, K.A.; Tyanova, S.; Herbert, A.S.; et al. 2018. Multiplexed proteome analysis with neutron-encoded stable isotope labeling in cells and mice. Nat Protoc, 3(1), 293-306. PMID: 29323663
Cox, A.G.; Hwang, K.L.; Brown, K.K.; et al. 2016. Yap reprograms glutamine metabolism to increase nucleotide biosynthesis and enable liver growth. Nat Cell Biol, 18(8), 886-896. PMID: 27428308
Baughman, J.M.; Rose, C.M.; Koluman, G. 2016. NeuCode proteomics reveals Bap1 regulation of metabolism. Cell Rep,16(2), 583-595. PMID: 27373151
McClatchy, D.B.; Ma, Y.; Liu, C. 2015. Pulsed azidohomoalanine labeling in mammals (PALM) detects changes in liver-specific LKB1 knockout mice. J Proteome Res, 14(11), 4815-4822. PMID: 26445171
Zhang, A.; Uaesoontrachoon, K.; Shaughnessy, C. 2015. The use of urinary and kidney SILAM proteomics to monitor kidney response to high dose morpholino oligonucleotides in the mdx mouse. Toxicol Rep, 2, 838-849. PMID: 26213685
Hathout, Y.; Marathi, R.L.; Rayavarapu. 2014. Discovery of serum protein biomarkers in the mdx mouse model and cross-species comparison to Duchenne muscular dystrophy patients. Hum Mol Genet, 23(24), 6458-6469. PMID: 25027324
Schiapparelli, L.M.; McClatchy, D.B.; Liu, H.H. 2014. Direct detection of biotinylated proteins by mass spectrometry. J Proteome Res, 13(9), 3966-3978. PMID: 25117199
Rayavarapu, S.; Coley, W.; et al. 2013. Activation of the ubiquitin proteasome pathway in a mouse model of inflammatory myopathy: a potential therapeutic target. Arthitis Rheum, 65(12), 3248-3258. PMID: 24022788
Andrew Percy, PhD
Senior Applications Chemist – Mass Spectrometry
Dr. Andrew Percy is the Senior Applications Chemist for Mass Spectrometry and the MS ‘Omics Product Manager at CIL. His responsibilities minimally involve providing technical support, overseeing product development, identifying new product market opportunities, assisting in the analysis of product-related applications, and writing/reviewing marketing literature.
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Kevin Millis, PhD
Senior Scientist, Application Development Manager
Kevin Millis, PhD, is the Senior Scientist and Market Development Manager for all NMR and mass spectrometry product lines. Kevin is responsible for Technical Services both internally and externally for all CIL customers as well as being responsible for the application and market development for the CIL products.
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