Procedure summary for MS experiments using SILAC reagents. Normalized protein extracts isolated from cells are combined, reduced, alkylated, and digested overnight. For the in-gel workflow, samples are run on an SDS-PAGE gel, excised, digested, and cleaned up; for the in-solution workflow, samples are digested, fractionated, and cleaned up. Samples are then analyzed by high-resolution Orbitrap LC-MS/MS.
SILAC Metabolic Labeling Systems
Stable isotope labeling using amino acids in cell culture (SILAC) is a powerful method to identify and quantify relative differential changes in complex protein samples. The SILAC method uses in vivo metabolic incorporation of “heavy” 13C- or 15N-labeled amino acids into proteins followed by mass spectrometry (MS) analysis for accelerated comprehensive identification, characterization and quantitation of proteins. NeuCode amino acids enable up to four samples to be multiplexed simultaneously.
- Reproducible—eliminates intra-experimental variability caused by differential sample preparation
- Flexible—media deficient in both L-lysine and L-arginine, as well as Flex media formulations that allow the addition of glucose, glutamine and phenol red separately
- Versatile—broadest portfolio of liquid and powdered SILAC media, as well as unique NeuCode amino acids, enabling higher multiplexing
- Convenient—media and amino acids are available in kits or as stand-alone reagents
- Compatible—label proteins expressed in a wide variety of mammalian cell lines, including HeLa, 293T, COS7, U2OS, A549, NIH 3T3, Jurkat and others
How-to video
SILAC metabolic labeling using mass spectromety
Learn how to prepare and metabolically label cultured cells using standard heavy or NeuCode amino acids for SILAC applications using mass spectrometry.
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Representative MS spectra generated using SILAC. Light and heavy (13C6) L-lysine-containing peptides (AEDNADTLALVFEAPNQEK) from PCNA were analyzed by MS. Mass spectra of heavy peptides containing 13C6 L-lysine have an increased mass of 6Da and are shifted to the right of light peptide spectra by a mass to charge ratio (m/z) of 3 caused by a +2 ionization of peptides
Table 1. SILAC isotopes of amino acids available to enable multiplexed experiments and analysis.
| Amino acid | Light | D4 | 13C6 | D8 | 13C15N2 | 13C615N4 |
|---|---|---|---|---|---|---|
| Mass shift | 0 Da | +4 Da | +6 Da |
+8 Da | +8 Da | +10 Da |
| L-Arginine-HCl | Cat.No. 89989 88427 (500 mg) |
N/A | Cat.No. 88210 (50 mg) 88433 (500 mg) |
N/A | N/A | Cat.No. 89990 (50 mg) 88434 (500 mg) |
| L-Leucine | 88428 (500 mg) | N/A | 88435 (50 mg) 88436 (500 mg) |
N/A | N/A | N/A |
| L-Lysine-2HCl | 89987 (50 mg) 88429 (500 mg) |
88437 (50 mg) 88438 (500 mg) |
89988 (50 mg) 88431 (500 mg) |
A33613 (50 mg) A33614 (500 mg) |
88209 (50 mg) 88432 (500 mg) |
N/A |
| L-Proline | 88211 (115 mg) 88430 (500 mg) |
N/A | N/A | N/A | N/A | N/A |
Table 2. Thermo Scientific NeuCode isotopes of L-lysine-2HCl available to enable multiplex experiments and analysis.
| +4 Da | +8 Da | ||||||
|---|---|---|---|---|---|---|---|
| Amino acid | K202 | K040 | K080 | K521 | K341 | K440 | K602 |
| Mass shift | 4.00078 | 4.02511 | 8.01420 | 8.02637 | 8.03221 | 8.03853 | 8.05021 |
| Cat. No. | A36754 (25 mg) | 88437 (50 mg) 88438 (500 mg) |
A36750 (25 mg) A33613 (50 mg) A33614 (500 mg) |
A36753 (25 mg) | A36851 (25 mg) | A36752 (25 mg) | A36751 (25 mg) 88209 (50 mg) 88432 (500 mg) |
Different combination of lysine isotopologs can be used to increase multiplexing from 2-plex to 4-plex at high resolution.
Click to enlarge
Procedure summary for MS experiments using SILAC reagents. Normalized protein extracts isolated from cells are combined, reduced, alkylated, and digested overnight. For the in-gel workflow, samples are run on an SDS-PAGE gel, excised, digested, and cleaned up; for the in-solution workflow, samples are digested, fractionated, and cleaned up. Samples are then analyzed by high-resolution Orbitrap LC-MS/MS.
Representative MS spectra generated using SILAC. Light and heavy (13C6) L-lysine-containing peptides (AEDNADTLALVFEAPNQEK) from PCNA were analyzed by MS. Mass spectra of heavy peptides containing 13C6 L-lysine have an increased mass of 6Da and are shifted to the right of light peptide spectra by a mass to charge ratio (m/z) of 3 caused by a +2 ionization of peptides
Table 1. SILAC isotopes of amino acids available to enable multiplexed experiments and analysis.
| Amino acid | Light | D4 | 13C6 | D8 | 13C15N2 | 13C615N4 |
|---|---|---|---|---|---|---|
| Mass shift | 0 Da | +4 Da | +6 Da |
+8 Da | +8 Da | +10 Da |
| L-Arginine-HCl | Cat.No. 89989 88427 (500 mg) |
N/A | Cat.No. 88210 (50 mg) 88433 (500 mg) |
N/A | N/A | Cat.No. 89990 (50 mg) 88434 (500 mg) |
| L-Leucine | 88428 (500 mg) | N/A | 88435 (50 mg) 88436 (500 mg) |
N/A | N/A | N/A |
| L-Lysine-2HCl | 89987 (50 mg) 88429 (500 mg) |
88437 (50 mg) 88438 (500 mg) |
89988 (50 mg) 88431 (500 mg) |
A33613 (50 mg) A33614 (500 mg) |
88209 (50 mg) 88432 (500 mg) |
N/A |
| L-Proline | 88211 (115 mg) 88430 (500 mg) |
N/A | N/A | N/A | N/A | N/A |
Table 2. Thermo Scientific NeuCode isotopes of L-lysine-2HCl available to enable multiplex experiments and analysis.
| +4 Da | +8 Da | ||||||
|---|---|---|---|---|---|---|---|
| Amino acid | K202 | K040 | K080 | K521 | K341 | K440 | K602 |
| Mass shift | 4.00078 | 4.02511 | 8.01420 | 8.02637 | 8.03221 | 8.03853 | 8.05021 |
| Cat. No. | A36754 (25 mg) | 88437 (50 mg) 88438 (500 mg) |
A36750 (25 mg) A33613 (50 mg) A33614 (500 mg) |
A36753 (25 mg) | A36851 (25 mg) | A36752 (25 mg) | A36751 (25 mg) 88209 (50 mg) 88432 (500 mg) |
Different combination of lysine isotopologs can be used to increase multiplexing from 2-plex to 4-plex at high resolution.
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