Expressing PP1 isoforms as GFP-tagged proteins in mammalian cells offers us both the opportunity to compare their dynamic localizations within live cells and the ability to recover the fusion proteins from cell lysates or purified structures and analyze the proteins with which they associate. This type of approach should identify both targeting subunits (proteins that interact directly with PP1) and other proteins that are in larger complexes with PP1 but do not interact with the phosphatase directly. These studies are carried out in collaboration with Jens Andersen and Matthias Mann at the Center for Experimental Bioinformatics in Odense, Denmark. The basic steps in the proteomic analyses include purification of nuclei (or other subcellular structures) from cultured mammalian cells stably expressing either GFP alone or a GFP-PP1 fusion protein, immunoprecipitation of these GFP proteins using anti-GFP antibodies, separation of the proteins by 1-dimensional polyacrylamide gel electrophoresis and LC/MS separation, identification of the proteins by peptide mass fingerprinting and mass spectrometry and bioinformatic analyses (cross-reference of protein informatics with genomic databases). Identified proteins are then further characterized by expression in mammalian and bacterial cells, generation of antibodies, etc. For more information about nuclear substructure and purification of subnuclear bodies, visit the Lamond lab website. For more information about mass spectrometry, visit the Mann lab website and the Center for Experimental Bioinformatics. Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) Standard proteomic approaches tend to produce large lists of proteins, some of which are specific to the purified complex or organelle of interest but many of which are contaminants. In order to quickly identify "real" hits and rule out contaminants, and to determine whether a particular PP1 binding partner showed specificity for either of the two isoforms we were studying (PP1α or PP1γ), we chose to use the SILAC approach (1,2) developed by our collaborators at CEBI.   Three populations of a cell line (or three different cell lines) can be metabolically labeled with either normal arginine (12C6-Arg, also referred to as Arg0), carbon-substituted arginine (13C6-Arg, also referred to as Arg6) or carbon- plus nitrogen-substituted arginine (13C615N4-Arg, also referred to as Arg10) respectively, for at least five cell doublings. This ‘triple encoding’ procedure allows three cell states to be measured in one experiment (3). The cells are identical in all respects except that peptides derived after proteolytic digestions of the proteins can be distinguished in the mass spectrometer by their offsets of either zero, six or ten mass units. For more information about SILAC, visit the SILAC page at CEBI. 1. S. E. Ong et al., Mol Cell Proteomics 1, 376-86. (2002). 2. S. E. Ong, I. Kratchmarova, M. Mann, J Proteome Res 2, 173-81 (2003). 3. B. Blagoev, S. E. Ong, I. Kratchmarova, M. Mann, Nature Biotech 22, 1139-45 (2004). Identifying nuclear interacting partners for PP1&alpha and PP1&gamma Nuclear lysates were prepared from all 3 cell lines, total protein concentration measured and the lysates combined in equal amounts. All three GFP proteins (GFP alone and the two PP1 fusions) were immunoprecipitated together from this combined lysate, using anti-GFP antibodies covalently conjugated to protein G sepharose. The immunoprecipitates were then analyzed by mass spectrometry to identify all the proteins that came down with the GFP fusion proteins, and the ratio of normal : heavy : very heavy arginine calculated for each. Schematic of the SILAC approach used to compare proteins that co-immunoprecipitated with either GFP alone, GFP-PP1α or GFP-PP1&gamma, or some combination of these three fusion proteins. protein comes down with GFP only (CONTAMINANT) protein comes down with GFP as well as the GFP fusion proteins (CONTAMINANT) protein comes down with GFP-PP1&alpha only (REAL HIT; targeting subunit for specific isoform) protein comes with with GFP-PP1&gamma only (REAL HIT; targeting subunit for specific isoform) protein comes down with both GFP-PP1&alpha and GFP-PP1&gamma (REAL HIT; targeting subunit associates with both isoforms. Can calculate ratio to determine if it shows a preference for either of them)
An example of a dataset obtained in this way is shown below. The ratio of heavy to normal arginine was calculated (GFP-PP1&alpha:GFP), so that we could identify PP1&alpha-specific binding proteins. These bars are plotted as positive numbers. The ratio of very heavy to normal arginine was also calculated (GFP-PP1&gamma:GFP), so that we could identify PP1&gamma-specific binding proteins. These bars are plotted as negative numbers, so that they can be compared to the PP1&alpha data.
	Preference for PP1&alpha
	Preference for PP1γ

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.

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, 2006.

		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).
Examples of peptide spectra from our GFP vs. GFP-PP1alpha vs. GFP-PP1gamma SILAC IP screen:

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.

Quantify the arginine and lysine ratios (heavy:light) for each peptide and plot on a graph:

The real hits (ratios > 1) are clearly identifiable over the background of contaminants (ratio = 1).

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.

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:

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.

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.