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Mass
spectrometry-based proteomics has become a powerful tool for identifying and
quantifying the components of multiprotein complexes. More recently, several
techniques have exploited the use of heavy isotope tags to compare and
quantitate relative protein levels under different biological conditions. In
the case of stable isotope labeling of amino acids in cell culture (SILAC),
cells are metabolically labeled through growth in medium containing specific
amino acids with either carbon or nitrogen or both substituted with the heavy
isotopes 13C and 15N. By using substituted arginine and/or lysine, proteins
are labeled specifically at sites of trypsin cleavage, which is convenient
for subsequent analysis of tryptic peptides by mass spectrometry.
Recently, we adapted the SILAC approach to identify interacting proteins that
co-immunopurify with our tagged protein of interest, incorporating a negative
control (cells expressing the tag alone) to rapidly identify real hits above
a background of contaminants. These contaminants can be environmental (e.g.
keratins), bind nonspecifically to the beads or the antibody, or associate
with the tag itself. The main strength of this approach is that the
immunoprecipitations can be done under less stringent conditions, preserving
both high and low affinity interactions. Although the number of contaminants
may increase under these condition, they can be easily dentified because they
are copurified in equal amounts from both the cells expressing the tag alone
and those expressing the tagged protein (and hence have a ratio of
"heavy" to "light" amino acids of 1:1). Interacting
proteins that are present in low abundance can also be identified more
readily using this technique, as their SILAC ratios clearly identify them as
real hits.
Publication:
 "Repo-Man
recruits PP1γ to chromatin and is essential for cell viability"
(2006) Trinkle-Mulcahy, L., Andersen, J., Lam, Y.W., Moorhead, G., Mann, M.
and Lamond, A.I. . J. Cell
Biol. 172:679-92.
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When cells are grown in media
containing differentially labeled isotopes of an essential amino acid such
as arginine, all the proteins incorporate the specific amino acid.
If you combine cells from all three dishes, tryptic peptides for a
particular protein will thus exist as three forms: light (from the R0
dish), heavy (from the R6 dish) and very heavy (from the R10 dish). In the
case of labeled arginine, the peaks are due to a mass shift of either 6 Da
(R6) or 10 Da (R10) from the standard form (R0).
If the protein is found in equal amounts in all three of your experimental
conditions, the ratio of these three peaks will be 1:1:1, as shown here.
If the protein is enriched in one of your conditions, it will be seen as an
increase in that particular peak relative to the others (see below).
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Examples of peptide spectra from our GFP vs. GFP-PP1alpha vs. GFP-PP1gamma
SILAC IP screen:
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Double Encoding SILAC
Experiment
This is commonly carried out when you wish to compare immunoprecipitation of
your tagged protein of interest to that of the tag alone. Contaminants will
either be environmental (e.g. keratins) or sticking nonspecifically to the
beads, antibody or tag. We now use a combination of labeled arginine and
labeled lysine to ensure that every tryptic peptide contains a quantifiable
amino acid (trypsin will cut after an arginine or lysine).
In this case the R0K0 media refers to media containing 12C-Arginine and
12C-Lysine. The R6K6 media contains 13C-Arginine and 13C-Lysine. For details
on sourcing reagents and preparing the media, download our SILAC Reagent Protocol.
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Quantify the arginine
and lysine ratios (heavy:light) for each peptide and plot on a graph:
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The real hits (ratios > 1) are clearly identifiable over the background
of contaminants (ratio = 1).
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Triple Encoding SILAC
Experiment
SILAC also offers the means to compare two different tagged proteins in the
same experiment, while retaining the internal negative control (tag alone).
This can be used to compare two different isoforms of the same protein (e.g.
PP1alpha vs. PP1gamma), a wild type and a mutant form of the same protein, a
wild type and a phosphomimic (or nonphosphorylatable) mutant of a protein,
etc. It can also be used to compare the same protein under two different
cellular conditions (e.g. untreated vs. DNA damaged or transcriptionally
inhibited).
In this case the R0K0 media refers to media containing 12C-Arginine and
12C-Lysine. The R6K4 media contains 13C-Arginine and 4D-Lysine. The R10K8
media contains 13C/15N-Arginine and 13C/15N-Lysine. For details on sourcing
reagents and preparing the media, download our SILAC
Reagent Protocol.
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Quantify the
arginine and lysine ratios (heavy:light) and (heavy/heavy:light) for each
peptide and plot on a graph.
For clarity, the
heavy:light ratios (protein 1:tag alone) are plotted as positive values
while the
heavy/heavy:light
ratios (protein2:tag alone) are plotted as negative values:
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The real hits (ratios > 1) are clearly identifiable over the background
of contaminants (ratio = 1),
and we can also clearly see which of these real hits shows a preferential
association with one of the proteins.
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SILAC Protocols:
For details on sourcing reagents and preparing the media, download our SILAC Reagent Protocol.
For details on covalently coupling antibodies to Protein G sepharose,
download our Covalent Coupling
Protocol.
For details on cell fractionation and preparation of whole cell vs. nuclear
and cytoplasmic lysates, download our Cell
Fractionation Protocol.
For details on trypsin digestion of gel slices for MS analysis, download our In Gel Digestion Protocol.
Check out our new SILAC FAQ.
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