Proteomics

SILAM

Stable Isotope Labeling in Mammals (SILAM)

John R. Yates, III, PhD
Ernest W. Hahn Professor
The Scripps Research Institute, Chemical Physiology & Cell Biology


  • Stable Isotope Labeling in Mammals (SILAM)
  • 15N Stable Isotope Labeling Data Analysis
  • Stable Isotope Labeling in Mammals with 15N Spirulina (Application Note 24)
  • Targeted LC-SRM/MS Quantification of Mammalian Synaptic Proteins with MouseExpress® Brain Tissue (Application Note 27)
 

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. CIL is also pleased to offer intact stable isotope-enriched mouse tissue for use in SILAM: Mouse Express® L-Lysine (13C6, 97%) Mouse Tissue and Mouse Express® (15N, 94%) Mouse Tissue. 

 

Resources

Feed for SILAM
Mouse Express® NeuCode™ Mouse Feed
L-Azidohomoalanine·HCl

 

 

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 the macronutrient information for a representative Spirulina 15N-containing diet?

Metabolic Labeling (SILAM) spirulina

What is the macronutrient information for an amino acid-defined diet lysine?

Metabolic Labeling (SILAM) amino acid

What is contained in the Mouse Express® Mouse Feed Kits?

  • Mouse Express® L-Lysine (13C6, 99%) Mouse Feed Kit, Irradiated Kit contains: 1 kg of L-Lysine 13C6 feed and 1 kg of unlabeled feed.
  • Mouse Express® (15N, 99%) Mouse Feed Kit prepared with spirulina Kit contains: 1 kg spirulina (15N, 99%) feed and 1 kg spirulina (unlabeled) feed
  • Mouse Express® L-Leucine (5,5,5-D3, 99%) Mouse Feed Kit, Irradiated Kit contains: 1 kg of L-Leucine D3 feed and 1 kg of unlabeled feed

 

What quality control is performed on Spirulina?

• Appearance

• GC/MS for Identification

• GC/MS for Isotopic Enrichment

• ICP Analysis for the Presence of Metals

• Nitrates / Nitrites

• Moisture, Protein,Fat,Fiber,Ash,Mineral Content

• Protein based on Amino Acid Content

• Protein from Kjeldahl nitrogen (KN)

• pH Analysis

• Brine Shrimp Toxicity Test

 

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.

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 protease for proteomic analysis.

 

References 

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

Shen, W.; Liu, H.H.; Schiapparelli, L. 2014. Acute synthesis of CPEB is required for plasticity of visual avoidance behaviour in Xenopus. Cell Rep, 6(4), 737-747. PMID: 24529705

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

Andrew Percy, PhD

Senior Applications Chemist – Mass Spectrometry

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Kevin Millis, PhD

Kevin Millis, PhD

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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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