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In-vitro SILAC CTAP with DAP

The SILAC approach is a powerful tool in quantitative proteomics of cell cultures.[1] Two cell populations are cultured in separate media, one containing unlabelled and one 13C/15N-labelled arginine or lysine. , The two cell populations are then combined for mass spectrometry analysis facilitating relative quantitation of a great number of proteins.

The SILAC method has limited applicability in multicellular contexts as cultures usually have to be grown in isolation. A new technique for the investigation of cell-cell interactions is the cell type-specific labelling using amino acid precursors (CTAP).[2] Instead of the essential amino acid itself, a stable isotope (SI)-labelled substrate or precursor is used. By overexpressing an enzyme that synthesizes the desired amino acid from its precursor, a metabolic incorporation of the label into a specific cell type is achieved.[3]

 

 

Silantes High Quality Components for
Cell Type-Specific Labelling using Amino Acid Precursors

Silantes provides SI-labelled diaminopimelic acid (DAP), which is a precursor for the SILAC amino acid lysine. By transfecting distinct cell populations with the gene encoding the enzyme diaminopimelate decarboxylase (DDC), these cells gain the ability to grow on DAP instead of lysine.

Silantes offers 13C-, 15N- and 13C15N-labelled DAP in powder form, which is produced by fermentation. We guarantee an isotopic enrichment of > 97 atom % and a chemical purity of > 95 %. The isotopic and chemical purities are confirmed by mass spectrometry and HPLC, respectively.

Product

Mass Shift

Quantity

Article Number

13C-labelled L-Diaminopimelic acid powder

7 Dalton

100mg

1g

220203900

220204100

15N-labelled L-Diaminopimelic acid powder

2 Dalton

100mg

1g

220303900

220304100

13C 15N-labelled L-Diaminopimelic acid powder

9 Dalton

100mg

1g

220603900

220604100

 

 

 

Applications of the CTAP Approach using SI-labelled DAP

In cell-of-origin secretome studies with lysine-auxotrophic cells, biomarker and cell-cell communication analysis can be performed in co-culture by metabolic labelling with DAP as a substrate (Figure 1).

Figure_1_Cell_type-specific_labelling_using_amino_acid_precursors_CTAP_approach_by_Gauthier_et_al._Eigene_Darstellung

Figure 1: Cell type-specific labelling using amino acid precursors (CTAP). Method illustration from Gauthier et al.2,3

 

Figure 2 shows an example of a cell-of-origin secretome study using the method of Gauthier et al..3 A mouse cell line modified to express DDC for growth on DAP together with a human cell line capable of growing on D-lysine (expressing lysine racemase) were grown in co-culture on labelled D-lysine and unlabelled DAP. Nearly all proteins secreted from both cell lines could be distinguished by the difference in label illustrating the aptitude of CTAP for multicellular analysis. Additional applications of these studies are possible with the new availability of SI-labelled DAP.

Figure_2_CTAP_approach_for_secreted_factors_LC-MSMS_analysis_of_proteins_Eigene_Darstellung

Figure 2: CTAP approach for secreted proteins. LC-MS/MS analysis of proteins in the supernatants of DDC-expressing mouse (3T3) and lyr-expressing human (MDA-MB-231) cells co-cultured in SI-labelled D-Lys (H = heavy) and unlabelled DAP (L = light). Illustration from Gauthier et al.3

 

 

[1] Ong, S. E., Blagoev, B., Kratchmarova, I., Kristensen, D. B., Steen, H., Pandey, A., Mann, M. (2002). Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics. Mol Cell Proteomics. 1(5):376-386.

[2] Gauthier, N., Sander, C., Miller, M., (2014), Cell selective proteome labeling, WO 2014039643.

[3] Gauthier, N. P., Soufi, B., Walkowicz, W. E., Pedicord, V. A., Mavrakis, K. J., Macek, B., Gin, D. Y., Sander, C. and Miller, M. L. (2013). Cell-selective labeling with amino acid precursors for proteomic studies of multicellular environments. Nat Methods 10(8), 768-773.

 


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Product no.: 220304100

Chemical formula: C7H1415N2O4

Isotopic enrichment > 97 atom %

Chemical purity > 95 %

$1,390.00 / g *

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Product no.: 220303900

Chemical formula: C7H1415N2O4

Isotopic enrichment > 97 atom %

Chemical purity > 95 %

$290.00 / 100 mg *

In stock.
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Product no.: 220204100

Chemical formula: 13C7H14N2O4

Isotopic enrichment > 97 atom %

Chemical purity > 95 %

$3,990.00 / g *

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Product no.: 220203900

Chemical formula: 13C7H14N2O4

Isotopic enrichment > 97 atom %

Chemical purity > 95 %

$800.00 / 100 mg *

In stock.
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Product no.: 220603900

Chemical formula: 13C7H1415N2O4

Isotopic enrichment > 97 atom %

Chemical purity > 95 %

$970.00 / 100 mg *

Please contact our customer service for information on availability and delivery time at
webshop@silantes.com

Product no.: 220604100

Chemical formula: 13C7H1415N2O4

Isotopic enrichment > 97 atom %

Chemical purity > 95 %

$4,790.00 / g *

Please contact our customer service for information on availability and delivery time at
webshop@silantes.com

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