SILAC Media

Found in this section:

• SILAC Amino Acids
• Use of dialyzed serum
• Conversion of 13C6 Arginine to 13C5 Proline
• Common Media Formulations (DMEM and RPMI)
• Potentially useful SILAC amino acid stock solution table
• Commercial vendors

 

 

SILAC AMINO ACIDS

A variety of amino acids are suitable in SILAC and the use of arginine, leucine, lysine, serine, methionine and tyrosine have already been described in literature by several groups.  The use of an essential amino acid that does not metabolize to a different amino acid is most desirable in order to avoid a mixture of labelled amino acid products (See discussion on arginine conversion). 

Ideally, the amino acid used in SILAC should be able to introduce a large enough mass difference from the unlabeled peptide so that the two peak clusters can be easily distinguished.  

 

 

The comparison of the two amino acids we have described for use with SILAC is presented in the table above.  We favor the use of ¹³C-containing amino acids for use with liquid chromatography-mass spectrometric analyses.   We also like the use of arginine and lysine as labeling amino acids as most tryptic peptides contain one of each of these residues in the C-terminal position.  This also provides an added benefit for the determination of fragmentation ion species in tandem MS spectra too (Ong et. al. JPR 2003).

 

DIALYZED SERUM

As described in Ong et. al. MCP 2002, the use of dialyzed serum in culture medium formulation is necessary to avoid the contribution of natural stable isotope abundance amino acids in the serum in "heavy" labeled cell culture. 

 

This shows the situation where using normal foetal bovine serum (middle panel) results in a large mixture of peptides that contain a mixture of Leu-d0 and Leu-d3.  The peptide shown contains 3 leucines in total.  The peak cluster labeled with an asterix is an unrelated peptide.  Figure from Ong et. al MCP 2002.

 

Using dialyzed serum is important for accurate quantitation, however we have also found that some cell types behave anomalously in dialyzed serum, i.e. do not attach even after an extended period.  These cases form the minority of our experience in using SILAC and we have not further troubleshooted these cases.  We provide a list of cell types that we have grown successfully in the basic SILAC media compositions described here.

Commercial sources for FBS are widely available and the option to dialyze serum in one's own laboratory exists.  A lower molecular weight cut-off may help to reduce the loss of small bioactive peptides and lipids.  It is critical to evaluate each batch of dialyzed serum for the residual traces of amino acids. 

 

Formation of ¹³C5 Proline from ¹³C6 Arginine

Our experiments with Arg-13C6 show that some cell lines (e.g. HeLa) convert ¹³C6-arginine to ¹³C5-proline.  This results in the formation of 2 distinct peak clusters for all proline-containing peptides in the labeled state.  The correct amount of the "heavy" state is therefore the sum of both the Arg-13C6 and the Arg-¹³C6 + Pro-¹³C5 peak. 

This can be accounted for in two straightforward ways:

1) to add the contribution from the Arg-¹³C6 + Pro-¹³C5 peak cluster along with the Arg-¹³C6 peak.

2) to empirically reduce the amount of arginine used in the labeling media (both light and heavy media should have the same formulation) and monitor for non-conversion of arginine to proline.

We prefer the latter approach because it simplifies the quantitation and amount of work required.  We find that determination of the amount of arginine to use for each separate cell line by a titration series is useful.  Additionally, as arginine in the ¹³C labeled form is more expensive, an added advantage to reducing the arginine used in the media is a reduction in costs.  As an example, in titration experiments with attached HeLa cells, we have found that lowering the arginine concentration used in DMEM to 17 - 21 mg/L reduces arginine to proline conversion to a point where the 13C5-proline containing peptide is not detectable in the mass spectrum.  Whilst supplementing the media with proline could potentially avert this conversion process, it is conceivable that the reverse conversion process to form unlabeled arginine may then be favored, thus requiring another titration.  This has not been investigated.   

 

SILAC MEDIA FORMULATION

The table listed below is an example formulation of DMEM high-glucose without glutamine (catalog no. 21969) in the Gibco-Invitrogen catalog. The SILAC DMEM we use is based on this DMEM formulation but any of your standard formulations that work with the cell lines currently in your laboratory can be adapted for labeling in SILAC. (for e.g. picking a completely different formulation and leaving out the SILAC amino acids, and re-supplementing these later)

COMPONENTS	Molecular Weight	Concentration (mg/L)	Molarity (mM)
Amino Acids
Glycine	75	30	0.4
L-Arginine hydrochloride	211	84	0.398
L-Cystine 2HCl	313	48	0.201
L-Histidine hydrochloride-H2O	210	42	0.2
L-Isoleucine	131	105	0.802
L-Leucine	131	105	0.802
L-Lysine hydrochloride	183	146	0.798
L-Methionine	149	30	0.201
L-Phenylalanine	165	66	0.4
L-Serine	105	42	0.4
L-Threonine	119	95	0.798
L-Tryptophan	204	16	0.0784
L-Tyrosine disodium salt dihydrate	261	72	0.398
L-Valine	117	94	0.803
Vitamins
Choline chloride	140	4	0.0286
D-Calcium pantothenate	477	4	0.00839
Folic Acid	441	4	0.00907
i-Inositol	180	7.2	0.04
Niacinamide	122	4	0.0328
Pyridoxine hydrochloride	206	4	0.0194
Riboflavin	376	0.4	0.00106
Thiamine hydrochloride	337	4	0.0119
Inorganic Salts
Calcium Chloride (CaCl2) (anhyd.)	111	264	1.8
Ferric Nitrate (Fe(NO3)3"9H2O)	404	0.1	0.000248
Magnesium Sulfate (MgSO4) (anhyd.)	120	0	0.814
Potassium Chloride (KCl)	75	400	5.33
Sodium Bicarbonate (NaHCO3)	84	3700	44.05
Sodium Chloride (NaCl)	58	6400	110.34
Sodium Phosphate monobasic (NaH2PO4-H2O)	138	141	0.906
Other Components
D-Glucose (Dextrose)	180	4500	25
Phenol Red	376.4	15	0.0399
Sodium Pyruvate	110	110	1

This table was extracted from the Gibco-Invitrogen website and is originally their formulation for Dulbecco's Modified Eagle Medium (D-MEM) (1X) liquid (high glucose) - Contains 4500 mg/L D-glucose and sodium pyruvate but no L-glutamine. Catalog Number: 10313021

 

Potentially useful SILAC amino acid stock solution table

A suggested way to standardize the way your laboratory's stock solutions for SILAC amino acids - here in Excel format.  (The concentrations are merely suggestions, you should check whether it's appropriate for your own media formulations).

 

Commercial vendors for SILAC materials

There exists a wide variety of commercial vendors for material suitable for SILAC and we aren't able to provide a comprehensive list here.   

SILAC MEDIA:  

Invitrogen custom formulations (custom orders requiring certain minimal volume orders, the most convenient because you can specify exact formulations which may be more specialized for your needs).  Their media inquiry page is here.

Sigma Cell Culture Powder DMEM and RPMI (lacks leucine, lysine, methionine, glutamine, sodium bicarbonate)

JRH Biosciences - For custom formulations. Not one we have used ourselves but used successfully and reported by another SILAC user (Dr. Kathy Wong, BTI, personal communication).

 

SILAC AMINO ACIDS:

Cambridge Isotope Laboratories 

Sigma-Isotec

 

SILAC DIALYSED SERUM:

Dialysed Foetal Bovine Serum - Invitrogen

 

NORMAL CELL CULTURE SUPPLEMENTS:

Antibiotics 100x, Glutamine 100x - Invitrogen

 

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