Quantitative proteomics to study mitogen-activated protein kinases

Methods 40 (2006) 243–250 www.elsevier.com/locate/ymeth

Quantitative proteomics to study mitogen-activated protein kinases Blagoy Blagoev a,¤, Matthias Mann a,b,¤ a

Center for Experimental Bioinformatics (CEBI), Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark b Department of Proteomics and Signal Transduction, Max-Planck-Institute of Biochemistry, D-82152 Martinsried, Germany Accepted 10 August 2006

Abstract In the last several years, the impact of mass spectrometry (MS)-based proteomics on cell signaling research has increased dramatically. This development has been driven both by better instrumentation and by the progression of proteomics from mainly qualitative measurements towards quantitative analyses. In this regard, Stable Isotope Labeling by Amino acids in Cell culture (SILAC) has established itself as one of the most popular and useful quantitative proteomic methodologies to study signaling networks. SILAC relies on the metabolic incorporation of non-radioactive heavy isotopes in the whole proteome of desired cell line, making all proteins from these cells easily distinguishable in the mass spectrometers from the proteins originating from control cells. The procedure does not involve any chemical derivatization steps and, importantly, allows mixing of the two cell populations for combined additional sample manipulation, thus leading to highly reliable results with minimal errors. In this chapter, we describe in detail the SILAC labeling procedure and explain how to design SILAC experiments to examine the level and duration of phosphorylation of endogenous MAP kinases and their substrates in cell culture systems. © 2006 Elsevier Inc. All rights reserved. Keywords: Protein quantitation; SILAC; Mass spectrometry; Phosphorylation; Stable isotope labeling

1. Introduction In the post-genomic era, high throughput mass spectrometry (MS) has become capable of identifying thousands of proteins from biological samples. Such large-scale analysis supplied the Wrst useful proteome maps of distinct cellular organelles, various tissues and body Xuids, and even small organisms [1–8]. However, these valuable studies provide only “snap-shots” of the investigated structures and were unable to unveil the dynamic nature of complex biological systems. The rapid development of quantitative MS-based proteomics techniques in recent years (reviewed in [9]) added a new dimension to proteomic research—the ability to follow quantitative changes of the proteomes in * Corresponding authors. Fax: +45 6593 3018 (B. Blagoev), +49 89 8578 2219 (M. Mann). E-mail addresses: [email protected] (B. Blagoev), mmann@biochem. mpg.de (M. Mann).

1046-2023/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.ymeth.2006.08.001

space and time [10,11]. Quantitative proteomics facilitates determination of even subtle changes in protein expression levels or post-translational modiWcations (PTMs) as a result of a drug treatment, changes in the cellular environment or alterations in the total body homeostasis [9]. It also makes possible to investigate combinatorial signaling events and interplay of diVerent pathways involving hundreds of proteins and to identify controlling points leading to a deWned cellular outcome [12]. Today, quantitative MSbased proteomics is the method of choice for high throughput determination of high conWdence data required for a global understanding of the spatial and temporal order of regulatory events that take place in a cell. Stable isotope labeling by amino acids in cell culture (SILAC) is a powerful and relatively simple method for an accurate quantitation of proteins by mass spectrometry [13,14]. It employs heavy amino acids containing nonradioactive stable isotopes that are metabolically incorporated into the newly synthesized proteins of the cells.

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Through the SILAC-labeling procedure the entire proteomes of given cellular populations become encoded either with the light or heavier version of the same amino acid enabling their direct quantitative comparison using mass spectrometry. After encoding the “light” and “heavy” cells, these are either mixed directly or lysed in a desired buVer and then the cell lysates are mixed followed by optional protein puriWcation, fractionation, interaction assay, etc. When performed separately for the two samples, each of these steps contributes its own variability, which signiWcantly decreases the overall accuracy of the measurement. The possibility of combining the SILAC samples at an early stage of the experiment constitutes a critical advantage compared to other quantitative proteomics approaches as it results in minimized quantitation errors [9]. In addition, SILAC does not involve any sophisticated chemistry; it is an easily applicable and non-demanding method that can be used in a broad range of cell culture models. Due to its intrinsic advantages SILAC is becoming a quantitative proteomics method of choice. It has already been successfully employed to study a large variety of biological systems. One of the obvious applications of SILAC is to monitor expression changes, in a way similar to cDNA microarray-type experiments, but directly at the protein level. Indeed, SILAC was originally used to quantitate changes in the protein expression levels during the process of muscle cell diVerentiation [14]. However, the major strength of the SILAC approach is that it can be used to distinguish speciWc from non-speciWc protein interactions, to quantify regulatory post-translational proteins modiWcations and to characterize functional protein complexes. A study describing 28 proteins associated with the phosphorylated epidermal growth factor receptor (EGFR) provides a prototypical example [15]. In this work, SILAC allowed these 28 proteins to be distinguished as speciWcally enriched in the activated EGFR complex among a large background of over 200 proteins identiWed in the pull down. Used in a similar manner, SILAC has found many applications including characterization of the phosphotyrosine interactome of the ErbB-receptor family, the protein phosphatase 1 alpha and gamma complexes and the 26 S proteasome [16–18]. Since biological systems are not static but highly dynamic in their response to stimuli, the investigation of cell signaling cascades and creation of protein interaction networks represents one of the largest challenges in the Weld of system biology. It is of critical importance for the comprehensive understanding of any biological process to follow its development over time. In this regard, instead of frozen image of the events occurring after addition of growth factors, SILAC was used to create a dynamic map of the entire phosphotyrosine-dependent signaling network induced by EGF, comprised of 81 proteins [11]. In this investigation, several members of the MAPK family were identiWed as well. Notably, the activation of both ERK1 and ERK2 by EGF was more rapid and transient com-

pared to a delayed and prolonged activation proWle of the p38 MAPK. These three MAPKs also appeared in another SILAC study comparing the signaling networks initiated by EGF and platelet-derived growth factor (PDGF) in mesenchymal stem cells [12]. The quantitative proteomics strategy revealed that 90% of the activated proteins in these cells were utilized by both ligands. Among those, ERK1/2 and p38 MAPKs were stimulated to very similar levels both by EGF and PDGF and their activity were found essential for osteoblasts diVerentiation. Not surprisingly, being the major downstream eVectors of a broad range of stimuli, various members of MAPK cascades were identiWed in diverse quantitative proteomics analyses including insulin, EphB2, FGF and T-cell receptor signaling cascades and yeast pheromone response pathway [19–23]. Importantly, global quantitative proteomics approach like SILAC do not just identify diVerent MAPKs in a speciWc signaling pathway but also put them into an upstream and downstream context, helping to determine their regulatory mechanisms or to provide direct and indirect targets and eVectors of the MAPK signaling cascades. What is needed for a cell signaling researcher wishing to use SILAC-based proteomics? The experimental requirements involving cell culture and signaling assays are relatively straightforward and are detailed below. They will not be a major obstacle. However, at this point, the researcher still has to obtain access to a proteomics facility as the mass spectrometers and associated technology are still relatively specialized and expensive. The mass spectrometric expertise needed varies with the type of experiment. At the one extreme, SILAC can be used to quantify phosphorylation status of a single puriWed protein between control and stimulus. Obtaining this data and interpreting it are usually straightforward for any MS unit. At the other extreme, “reading out” the entire phosphoproteome of a cell line as a function of time is currently a major undertaking involving many weeks of work even for the most advanced and specialized laboratories. However, we expect SILAC-based quantitative proteomics to become a standard assay over the next few years. 2. Materials 2.1. Cell culture for SILAC Labeling 1. Media: • Dulbecco’s ModiWed Eagle Medium (DMEM) deWcient in lysine and arginine described here for culturing adherent HeLa cells (see Note 1). • RPMI-1640 deWcient in lysine and arginine given as an example here for culturing suspension HeLa S3 cells (see Note 1). 2. Amino acids: • Normal “light” amino acids: L-lysine and L-arginine hydrochloride (referred later in the text as Lys0 and Arg0, respectively) (Sigma Chemicals, Copenhagen, Denmark).

B. Blagoev, M. Mann / Methods 40 (2006) 243–250

• Stable isotope-labeled “heavy” amino acids: L-arginine-13C6 hydrochloride (Arg6) (Cambridge Isotope Labs, Andover, MA; cat. no. CLM-2265), L-arginine13 C6, 15N4 hydrochloride (Arg10) (cat. no. 608033), Llysine-4,4,5,5-d4 hydrochloride (Lys4) (cat. no. 616192), and L-lysine-13C6, 15N2 hydrochloride (Lys8) (cat. no. 608041) (Sigma-Isotec, St. Louis, MO). 3. Supplements: • Dialyzed Fetal Bovine Serum (FBS) (Gibco-Invitrogen, Carlsbad, CA). • L-Glutamine (200 mM stock solution) (Gibco-Invitrogen, Carlsbad, CA). • Penicillin/Streptomycin (10.000 U/10.000 g stock solution). 4. Additional: • Trypsin–EDTA solution (Trypsin, 200 mg/L and VerseneEDTA 500 mg/L) (Gibco-Invitrogen, Carlsbad, CA). • Sterile phosphate buVered saline (PBS) (Cambrex Bio Science Copenhagen ApS, Denmark). • Filter units, MF75TM series (Nalge Nunc International, NY). 5. Cell lines: • Adherent HeLa cells, epithelial adenocarcinoma (American Type Culture Collection (ATCC), Manassas, VA). • Suspension HeLa S3 cells, epithelial adenocarcinoma (American Type Culture Collection (ATCC), Manassas, VA).

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7. Colloidal Blue Stain (Invitrogen) for visualizing proteins on the gel. 8. Sequence grade-modiWed trypsin (Promega, Madison, WI) for the proteolytic digestion of the proteins from gel slices. 3. Methods 3.1. SILAC media preparation

2.2. Cell lysis, aYnity puriWcation, and gel electrophoresis

The main requirement for applying SILAC to any cellular system is the ability of the cells to grow in the labeling medium for at least Wve cell doublings, essential for incorporation of the label (see Notes 2 and 3). A schematic presentation of the SILAC-labeling procedure is outlined in Fig. 1. In the following protocol, we describe the preparation of DMEM-based SILAC medium for encoding adherent HeLa cells with 13C6-arginine, however, any medium with deWned formulation can be used (see Notes 1–5). In such cases, the amino acids that will be used for the labeling should be left out of the original formulation. Since dialyzed serum is utilized throughout the labeling procedure (see Note 3), the only source of these essential amino acids for the cells become the “light” or “heavy” SILAC amino acids that are supplemented into the medium. This Xexibility has allowed the SILAC approach to be successfully extended not only to various mammalian cell lines but also to bacteria, yeast, plants and – via a labeled cell line used as an internal standard – even to quantitate tissue samples [24–28].

1. Lysis buVer: • ModiWed RIPA: 50 mM Tris, pH 7.5, 150 mM sodium chloride, 0.25% sodium deoxycholate (Sigma–Aldrich, St. Louis, MO) and 1% NP-40 (Calbiochem, Merck, Darmstadt, Germany). • Protease inhibitors (Complete™ tablets, Roche Diagnostics, Mannheim, Germany). • Tyrosine phosphatases inhibitor sodium orthovanadate (1 mM; Sigma Chemicals, Copenhagen, Denmark), Serine/Threonine phosphatases inhibitors sodium Xuoride (5 mM; Merck, Darmstadt, Germany) and beta-glycerophosphate (5 mM; Sigma Chemicals). 2. Cell scrapers (Sarstedt, Newton, NC). 3. Coomassie plus – The Better Bradford™ Assay Reagent (Pierce, Rockford, IL) for estimating protein concentration of cell lysates. 4. Immobilized mouse monoclonal antibody against phospho-ERK1/2 (Thr-202 and Tyr-204) for the immunoprecipitation of the activated forms of the corresponding MAPKs (Cell Signaling Technology, Beverly, MA). 5. Dithiothreitol (DTT) and iodoacetamide (Sigma–Aldrich) for reduction and alkylation of proteins, respectively. 6. NuPAGE® Novex 10% Bis–Tris gel system with MOPS running buVer for polyacrylamide gel electrophoresis (Invitrogen).

1. Both the normal “light” L-arginine and L-lysine and the “heavy” (for example 13C6-arginine) SILAC amino acids are dissolved in phosphate-buVered saline (PBS). We recommend the preparation of a 1000£ stock solution based on the original DMEM formulation (see Note 6). These stocks can be stored at 4 °C for at least 3 months. Table 1 provides our recommendations for SILAC amino acids concentrations in the labeling media (see also Note 7). 2. For the “light” SILAC medium add 250 L L-arginine (Arg0) and 330 L L-lysine (Lys0) from the amino acid stock solutions to 1 L DMEM (deWcient in arginine and lysine). For the “heavy” add 250 L 13C6-arginine (Arg6) and 330 L L-lysine (Lys0) to 1 L DMEM (deWcient in arginine and lysine) (see Table 1 and Notes 1 and 5). In some cell lines (HeLa, for example), arginine might be metabolically converted to proline due to oversupply of arginine into the growth medium. For HeLa cells, we determined that using four times lower arginine concentration (21 mg/L) was suYcient to avoid this conversion without aVecting normal cell growth (see Note 7). 3. Filter both media using Xask Wlter units (Nalge Nunc) (see Note 6). 4. Supplement both media with 1% (v/v) Penicilin/Streptomycin and 1% (v/v) L-Glutamine (Gibco-Invitrogen).

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Intensity

Arg0 medium

Arg6 medium

Split into Arg0 and Arg6 labeling medium

Intensity

Cells growing in standard media

m/z

m/z

Intensity

Intensity

Passage cells in the corresponding medium for at least five cell doublings

+ 6 Da

m/z Intensity

m/z

After mixing of the differentially encoded cells relative protein abundance can be compared directly in the mass spectrum m/z

Fig. 1. SILAC labeling of cells. Growing cells in “light” or “heavy” SILAC medium results in metabolic incorporation of the corresponding “light” and “heavy” amino acids into the proteins. After Wve cell doublings entire proteomes of the two populations are diVerentially encoded, easily distinguished in the mass spectrum and can be quantitatively compared by MS analysis. Table 1 Concentrations of SILAC amino acids in the labeling medium Cell line with corresponding labeling media

Amino acid

Manufacturer’s amino acid concentration (mg/L)

Working SILAC amino acid concentration (mg/L)

Adherent HeLa in DMEM

Arginine Lysine

84 146

21 48

Suspension HeLa S3 in RPMI

Arginine Lysine

240 40

42 40

5. At this point save some “light” and “heavy” serum free medium, if required by the experimental design (for example for serum depravation of the cells prior to growth factor treatment). 6. Add 10% dialyzed fetal bovine serum (Gibco-Invitrogen) to both “light” and “heavy” media (see Note 3). The SILAC media are ready for use and can be stored at 4 °C. 3.2. Culturing cells in SILAC-labeling media Once the SILAC media is prepared, culturing cells in the labeling media does not require special materials, skills or instrumentation any diVerent from a normal cell culturing. 1. To begin the labeling procedure split one dish of HeLa cells growing in normal DMEM, into two new dishes — one containing “light” and the other – “heavy” SILAC medium (see Fig. 1). For this purpose aspirate the media from an approximately 80% conXuent dish, rinse the cells once with PBS and use 1 mL trypsin/EDTA solution to detach the cells. Transfer 300 L from the cell suspension

into a dish containing “light” SILAC medium and another 300 L to a dish with “heavy” SILAC medium (if performing a triple SILAC-labeling experiment (see Fig. 2 and Note 1), simply seed another 300 L into a dish containing the third SILAC label (for example Arg10). 2. Passage the cells in the respective SILAC-labeling medium and expand the culture into the desired number of dishes. A minimum of Wve cell doublings in the labeling media are needed to ensure complete incorporation of the label (see Note 2). The two cell pools are now diVerentially encoded and ready for further manipulations required by the experiment. 3.3. Treatment of cells, immunoprecipitation and gel electrophoresis In this paragraph, we provide an example of a general aYnity puriWcation procedure with triple encoding SILAC using Arg0, Arg6 and Arg10 (see Fig. 2). However, the outlined protocol is extremely Xexible. A wide-range of biological systems can be studied by following essen-

B. Blagoev, M. Mann / Methods 40 (2006) 243–250

Arg0

Stimulus 1

Stimulus 2

Arg6

Arg10

B Intensity

A Control

247

+ 6 Da

+ 4 Da

m/z Mix lysates 1:1:1

C

Relative Abundance

Digestion with trypsin

ERK 2 FRHENIIGINDIIR (3+)

100

Affinity purification with anti-phosphoERKs antibodies

80

60

40

20

Protein identification and quantitation by LC-MS

0 571

573

575

577

579

m/z

Fig. 2. Determination of MAPK activation by triple SILAC labeling. (A) Three populations of cells are diVerentially encoded with Arg0, Arg6 and Arg10, respectively. Following treatment with the corresponding stimuli, cell lysates are mixed in equal proportions and subjected to immunoprecipitation with anti-phosphoERKs antibodies. Precipitated complexes are separated by gel electrophoresis; protein bands containing ERKs are digested with trypsin and analyzed by mass spectrometry. (B) Distinguishing the arginine-containing peptides in the mass spectrum. Arg0 peptides (marked with gray circle) are separated by 6 Da from the Arg6 peptides (red circle) and by additional 4 Da from the Arg10 peptides (blue circle). (C) The ratios of the Arg6 (red circle) and Arg10 (blue circle) containing peptides over the Arg0 (grey circle) peptides reXect the degree of ERK activation by Stimulus 1 and 2, respectively. For example the peptide FRHENIIGINDIIR from ERK2 illustrates that Stimulus 1 causes eight fold stimulation of Erk2 and this activation is twice as strong compared to the Stimulus 2 treatment. (For interpretation of the references to colour in this Wgure legend, the reader is referred to the web version of this paper.)

tially the same schema shown in Fig. 2, but modifying some of the steps as required by the experimental aims (diVerent SILAC amino acids, stimuli, aYnity puriWcation, etc.). 1. When the SILAC-encoded cells reach approximately 80% conXuence, replace their media with the respective serum-free SILAC medium. For adherent HeLa cells, 12–14 h of serum deprivation is an optimal time frame. 2. Induce Arg6 and Arg10-labeled cells with the corresponding stimuli, while leaving the Arg0 cells nontreated (or mock-treated, if needed) to serve as a control. The treatment of the cells may range from various growth factors and chemical inhibitors to diVerent concentrations and duration of activation. 3. Remove all media from the culture dishes and scrape the cells in ice-cold lysis buVer containing both protease and phosphatase inhibitors. Incubate on ice for 15 min and clear the lysates from cell debris by centrifugation. Save a small aliquot from each lysate and freeze these down (see Notes 8 and 9). 4. Estimate the protein amounts in the lysates by Bradford assay and mix the three lysates one-to-one-to-one based on their measured concentration. Collect a small aliquot at this point and freeze it (see Notes 8 and 9).

5. To immunoprecipitate activated ERKs, add the immobilized anti-phosphoERK1/2 antibody into the mixed lysates and incubate with head-over-tail rotation for 4– 6 h at 4 °C (see Note 8). 6. Wash the beads three times with Wve volumes of ice-cold lysis buVer. Resuspend the beads in NuPAGE® sample buVer and elute precipitated complexes by heating at 70 °C for 10 min. Use 10 mM DTT and 55 mM iodoacetamide for the sequential reduction and alkylation of the proteins prior to gel electrophoresis. 7. Load the sample on the NuPAGE gel and stain with Colloidal Blue to visualize the proteins. 8. Excise the gel band spanning the area of 40–50 kDa as this region of the gel contains both ERK1 and ERK2. Digest the gel slice with trypsin and prepare the sample for MS analysis (not described in this chapter) [29–31]. In the MS, all peptides derived from ERK1/2 will display ratios reXecting the level of ERK activation by the corresponding stimulus. In other words, the fold changes in peptide intensities between Arg0 and Arg6, and Arg0 and Arg10, respectively, correspond to the fold activation of ERKs by the respective treatment (see Fig. 2C). Moreover, the triple SILAC also allows comparing directly the level of activation between the stimulus 1 and 2. As shown in Fig. 2C both treatments led to ERK2 activation, however, stimulus 1 triggered

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approximately twice as strong ERK2 response compared to stimulus 2. 3.4. Concluding remarks The procedures described above reveal only a small fraction of the broad range of SILAC applications. This useful quantitative proteomic approach is relatively simple yet provides the accurate measurements that are essential for the study of complex biological systems. In regard to MAPK signaling SILAC is not restricted only to the study of direct activation of various MAPKs in the way described in this chapter. The phosphorylation status of signaling pathways can be “read out” by SILAC in a similar manner but by additionally employing a perturbation of the network. For example, experiments can be done in cell lines derived from knock out animals or using RNAi silencing of speciWc signaling factors. SILAC is also the method of choice for determining true interaction partners of members of signaling pathways [15]. In this experiment, bait is exposed to encoded cell lysates and control is exposed to unlabeled lysates. Background proteins then have one to one SILAC ratios and speciWc binders have ratios signiWcantly diVerent from that value [17,32]. As mentioned in Section 1, SILAC can also measure the kinetics of entire signaling pathways. To achieve this, triple SILAC encoding is used to distinguish three cell populations, which are treated for three diVerent times [11]. Peptides from each phosphoprotein will appear as a triplet in the mass spectra and the relative intensity directly indicates the relative degree of activation at the corresponding time point. Multiplexing this experiment allows higher time resolution. Recently, we have extended this approach to the quantitation of individual phosphorylation sites and performed this experiment for the whole cell, resulting in kinetics of more than 6000 phosphorylation sites following growth factor stimulation [33]. In closing, we whish to emphasize that implementation of the latest innovations in the Weld of proteomics, ranging from sample preparation to instrumentation and software development, will further establish SILAC as a powerful technology and standard assay in system biology and drug–target discovery research.

2.

3.

4.

4. Notes 1. The procedure described above outlines the preparation of Arg0- and Arg6-labeling medium for a standard, double SILAC setup. In some cases, however, the experimental design might require diVerent SILAC labels to be used. Following essentially the same protocol but simply utilizing the desired SILAC amino acid(s), any variety of the labeling media can be prepared (see also Notes 4 and 5).For dynamic studies (multiple time points of stimulation, for example) or comparing several treatments (diVerent growth factors, various concentrations of inhibitors, etc.) we strongly recommend performing triple SILAC experiments [10–12]. This involves labeling

5.

three cell populations in parallel – one with Arg0, one with Arg6 and the last one with Arg10 (see Fig. 2). Measuring the three states simultaneously in the same MS run results in signiWcant improvement of the quantitation errors, which is a crucial requirement for an adequate comparison of multiple stimuli [11]. Moreover, in order to analyze three cell states with double SILAC one needs to perform two experiments with a common control point. This comparison can be easily achieved with one triple SILAC setup, also resulting in reduction in experimental cost. A minimum of Wve cell doublings are needed for complete incorporation of the SILAC amino acid into the entire proteomes of the cells. The labeling occurs trough protein synthesis and turnover during the normal processes of cell growth and cell division. After Wve cell doublings, even if a given protein does not undergo any degradation, the unlabeled protein will be diluted at least 32 times (2n, where n is the number of cell divisions). In other words, approximately 97% of the protein will be SILAC labeled in the worst case. Dialyzed serum should be used throughout the labeling procedure to achieve complete incorporation of the SILAC amino acids. The dialysis removes free amino acids from the serum but unfortunately small molecules such as some growth factors may also be lost. For this reason, particular cell types might need adaptation in SILAC media. If required, small amounts of undialyzed serum, in addition to the dialyzed serum, can be supplemented without signiWcantly aVecting quantitation accuracy [34]. The right selection of SILAC amino acids is of high importance for the success of the experiment. The mass diVerence between the “light” and “heavy” form should be at least 4 Da in order to provide suYcient separation of the SILAC peptide pairs in the mass spectra (see Fig. 1). Smaller mass diVerences may lead to inaccurate quantitation as a result of large overlaps of “light” and “heavy” peptide isotope clusters [13]. The type of stable isotope within the SILAC amino acid is also important for the accuracy of quantitation. We recommend using 13 C- and 15N-labeled amino acids (although more expensive) because deuterated (2H) peptides tend to elute earlier than their SILAC counterparts in the reverse-phase chromatography [13,35]. Lastly, arginine and lysine are good choices for labeling as trypsin cleaves C-terminal to these amino acids and trypsin is the most commonly used proteolytic enzyme in proteomics [29] (see also Note 1). Thus all tryptic peptides (except the C-terminal one of the protein) are labeled and furthermore, mass spectra are particularly easy to interpret. Ideally, for quantitative analyses of PTMs, every peptide after proteolytic digestion of the proteins should contain SILAC amino acid in order to be quantitable. For this purpose, using both arginine and lysine for labeling becomes very helpful [25,36]. Except for the carboxyl terminal peptide, essentially every tryptic peptide from a

B. Blagoev, M. Mann / Methods 40 (2006) 243–250

600.84

HSP 90 IDIIPNPQER (2+)

600.84 100

84 mg/L 13C -Arg 6

100

HSP 90 IDIIPNPQER (2+)

21 mg/L 13C -Arg 6

80

Relative Abundance

Relative Abundance

80

60

40

603.35

*

20

0 598

249

599

600

601

602

603

60

40

*

20

604

605

0 598

m/z

599

600

601

602

603

604

605

m/z

Fig. 3. Unnecessarily high amounts of arginine in the labeling media may result in its metabolic conversion into proline. For example, using 84 mg/L of 13 C6-arginine leads to formation of small amounts of 13C5-proline in HeLa cells. As a result, proline-containing peptides have a 5 Da heavier satellite peptide cluster (marked with ¤), possibly leading to quantitation errors for these peptides. Titrating arginine concentration to one-quarter (21 mg/L) greatly minimizes this conversion and signiWcantly reduces the experimental costs.

protein can now be quantiWed (see also Note 4). It is also possible to directly label the amino acids that are post-translationally modiWed. Examples include the use of methionine-(13C,2H3-methyl) for labeling methyl groups in several methylated residues [37] and tyrosine (13C9-tyrosine) for tyrosine phosphorylation studies [20]. 6. The stock solutions from the normal “light” amino acids can be prepared in large volumes (see Table 1 for concentrations) and sterile Wltered using 0.2 m syringe Wlter. As the “heavy” amino acids are usually provided in small quantities we do not Wlter their stock solution to avoid losses. The small mass diVerences of the “light” and “heavy” amino acids should also be accounted for when preparing the labeling media. For example, prepare Arg0 stock at 84 mg/mL whereas for the Arg6 stock use 86.4 mg/mL in order to achieve equal molar concentrations. 7. When labeling with Arg6, in some cell types the 13 C6-arginine can be metabolically converted to 13C5proline. By MS analysis, it is easy to monitor if such conversion occurs in the cell line of choice as the prolinecontaining peptides will have a 5 Da heavier satellite peptide cluster (see Fig. 3). In practice, the conversion of arginine to proline is not a signiWcant problem. It only occurs in some cell types and even in such cases there are several ways to overcome it. The easiest solution is simply to omit the quantitation of proline-containing peptides. However, we recommend titration of the arginine in the labeling media in order to determine the concentration leading to minimal proline conversion for any cell line (see Fig. 3). Moreover, the useable arginine concentration is not a precise value but rather a certain range, which makes it easier to establish. To reduce experimental costs, it is beneWcial to perform such dilution series tests even if the metabolic conversion does not appear in the cells of choice. For example, for labeling

adherent HeLa cells we use four times less arginine in the DMEM and for suspension HeLa we use six times less arginine in the RPMI media (see Table 1). Note that the same amounts of the corresponding forms of arginine should be added to both “light” and “heavy” SILAC media. 8. The amount of lysate required for detection of activated MAPKs depends on several factors, including their expression levels in the cells, the strength of activation by the stimuli and the sensitivity of the mass spectrometer used. As minimal amounts we recommend to use 7–8 mg protein lysate from each condition and 100 L antiphosphoERK1/2 antibody (50% slurry, Cell Signaling Technology) for immunoprecipitation. 9. Collecting samples (30–50 g) from the unmixed lysates is very useful. These could be used to check the level of incorporation of the “heavy” amino acids, in case of any troubleshooting. Saving a sample (30–50 g) from the mixed SILAC lysates is very important as well. In case of inaccurate Bradford measurements, this sample can be used as a quality control for checking the exact mixing ratio by mass spectrometry and calculating a correction factor. References [1] R. Aebersold, M. Mann, Nature 422 (2003) 198–207. [2] H. Steen, M. Mann, Nat. Rev. Mol. Cell. Biol. 5 (2004) 699–711. [3] M.P. Washburn, J.R. Yates 3rd, Curr. Opin. Microbiol. 3 (2000) 292– 297. [4] E. Lasonder, Y. Ishihama, J.S. Andersen, A.M. Vermunt, A. Pain, R.W. Sauerwein, W.M. Eling, N. Hall, A.P. Waters, H.G. Stunnenberg, M. Mann, Nature 419 (2002) 537–542. [5] Y. Ho, A. Gruhler, A. Heilbut, G.D. Bader, L. Moore, S.L. Adams, A. Millar, P. Taylor, K. Bennett, K. Boutilier, L. Yang, C. Wolting, I. Donaldson, S. SchandorV, J. Shewnarane, M. Vo, J. Taggart, M. Goudreault, B. Muskat, C. Alfarano, D. Dewar, Z. Lin, K. Michalickova, A.R. Willems, H. Sassi, P.A. Nielsen, K.J. Rasmussen, J.R. Andersen, L.E. Johansen, L.H. Hansen, H. Jespersen, A. Podtelejnikov, E. Niel-

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