Dissection of the insulin signaling pathway via quantitative phosphoproteomics

Krüger et al. 10.1073/pnas.0711713105.

Supporting Information

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SI Figure 4
SI Table 1
SI Table 2
SI Table 3
SI Table 4
SI Table 5
SI Figure 5
SI Table 6
SI Table 7
SI Figure 6
SI Figure 7
SI Table 8
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SI Table 10
SI Methods

SI Figure 4

Fig. 4. Estimation of the correction factor for protein mixing and quantitation error based on SILAC pairs. (a) SILAC pairs of »100 proteins from whole cell lysates from from a mixture of nonlabeled and labeled cells were quantified to estimate the mixing error between the two lysates. (b) The quantitation error s was calculated from the average of the relative standard deviation of all SILAC pairs. The red line indicates the resulting mixing error (a) and the average quantitation error of the s.d. (b).

SI Figure 5

Fig. 5. Workflow for determining the activation profile of insulin-induced changes of the phosphotyrosine phosphoproteome. Three cell populations were SILAC-labeled with natural and stable isotope substituted arginine and lysine amino acids. Each cell population was stimulated with insulin for the indicated time. The experiment was repeated to generate a five time point kinetic. The combined lysates were enriched for tyrosine phosphorylated proteins by immunoprecipitation. After SDS/PAGE and in gel digestion, samples were analyzed by mass spectrometry. The overlap of peak intensities after the indicated time intervals resulted in a five time point activation profile.

SI Figure 6

Fig. 6. Selected mass spectra of pS388 of IRS-2. The left part shows the MS of the parent ion and the right part indicates the MS/MS fragmentation pattern of the phosphopeptide. The table summarizes the detected phosphosites of IRS-1 and IRS-2.

SI Figure 7

Fig. 7. Peptide pull down with two pY bait peptides from the PDZK11 protein with lysates from brown adipocytes.

SI Table 1

Table 1.

SI Methods

Preparation of the Triple-Labeling SILAC Media. Arginine- and lysine- free DMEM was divided into three equal portions, and 28 mg/liter of the three Arginine isotopes were added separately to make the Arg-0, Arg-6, and Arg-10 media, respectively. In addition, 48.7 mg/liter L-lysine, L-lysine- D₄, and L-lysine¹³C₆, ¹⁵N₂ were supplemented separately to the three lots containing Arg-0, Arg-6, and Arg-10. Finally, glutamine and antibiotics were added to the media with the full complement of amino acids (Arg-0/Lys-H4, Arg-6/Lys-D4, and Arg-10/Lys-8) and thereafter sterile-filtered (Millipore). The sterile media were separated into two portions for dialyzed serum-containing and serum free-media.

For double labeling SILAC media we used a combination of Arg-0, Lys-H4 for the "unlabeled" state and Arg-10, Lys-8 for the "heavy" state.

For the experiments with brown preadipocytes, cells were grown as described above for at least five cell divisions in the corresponding labeling medium. This ensured a complete labeling of the cells. After a 16h starvation period the cells were treated with insulin (1 mg/ml, Sigma) and IGF-1 (1 mg/ml, R&D). After hormone incubation for 5 min at 37°C, the media were removed and the cells were immediately lysed with ice-cold modified RIPA buffer.

Mass Spectrometric Analysis. After staining of the gel with the Colloidal Blue Staining Kit (Invitrogen) from the two lanes (mixture 1 of 0, 5, and 10 min and mixture 2 of 1, 5, and 20 min) evenly sized gel pieces were excised from the gel and processed for enhanced liquid chromatography-mass spectrometry (GeLC-MS). The gel pieces were subjected to in gel reduction and alkylation, followed by trypsin digestion as described in ref. 1.

Finally, peptides were extracted twice by adding an equal volume of 30% acetonitrile/0.3% trifluoraceticacid (TFA) in water to digest the mixture followed by a final extraction with 100% acetonitrile. Extracts were evaporated in a speedvac to remove acetonitrile and subsequently acidified with 0.5% TFA. Samples were desalted and concentrated with "STAGE" tips and resuspended in 5 ml of 0.5% acetic acid/1% TFA (2).

Reverse phase nano-LC-MS/MS was done by using an Agilent 1100 nanoflow LC system (Agilent technologies) using a cooled thermostated 96-well autosampler. The LC system was coupled to a 7-Tesla LTQ-FT instrument (Thermo Electron) equipped with a nanoeletrospray source (Proxeon). Chromatographic separation of peptides was performed in a 10-cm long 8-mm tip opening/75-mm inner diameter capillary needle (Proxeon). The column was custom-made with methanol slurry of reverse-phase ReproSil-Pur C18-AQ 3-mm resin (Dr. Maisch GmbH). The tryptic peptide mixtures were autosampled at a flowrate of 0.5 ml/min and then eluted with a linear gradient at a flow rate 0.25 ml/min. The mass spectrometers were operated in the data-dependent mode to automatically measure MS, MS/MS, and MS3 spectra (3). LTQ-FT full scan MS spectra were acquired with a resolution r = 50,000 at m/z 400. The three most intense ions were sequentially isolated for accurate mass measurements by a FTICR selected ion monitoring (SIM) scan. The SIM scans had a 15-Da mass range, with a resolution of 50,000 and a target accumulation value of 80,000. After the SIM scans the ions were fragmented in the linear ion trap using collision-induced dissociation at a target value of 10,000.

The following search parameters were used in all MASCOT searches: maximum of 2 missed trypsin cleavages, cysteine carbamidomethylation, methionine oxidation, pSTY, N-term protein acetyl, SILAC labels: Lys-D4, Lys-8, Arg-6, and Arg-10. The maximum error tolerance for MS scans was 10 ppm and 0.5 Da for MS/MS and MS3 scans, respectively. Only proteins were identified and quantified that had at least 2 ion scores >20. MSQuant was used to verify and quantify the resulting peptide pairs.

Immunoprecipitation and Western Blot Analysis. Immunoprecipitation was performed as described in ref. 4. Briefly, for the triple labeling experiment, differentiated brown adipocytes (cells of eight 15 cm dishes per condition) labeled with either Arg-0/Lys-H4, Arg-6/Lys-D4 or Arg-10/Lys-8 were treated with 1 mg/ml of insulin for 0, 5, and 10 min. In a second label experiment under the same conditions, adipocytes were stimulated for 1, 5, and 20 min, respectively. After insulin stimulation the cells were lysed with 700-ml modified RIPA buffer containing 1% Nonidet P-40, 0.1% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, and 50 mM Tris (pH 7.5), 1 mM sodium orthovanadate, 5 mM NaF, 5 mM beta-glycerophosphate, and protease inhibitors (Complete tablets, Roche Diagnostics) and were left on ice for 15 min. The lysates were centrifuged at 14,000g to pellet cellular debris. A Bradford assay was performed to determine protein concentrations of the supernatant followed by mixing the lysates of the three conditions 1:1:1 before the immnunoprecipitation step. The 5-min stimulated Arg-6/Lys-D4 situation served as a common reference to normalize the two experiments. Lysates from these cells were pooled and divided into two equal lots for mixing with the 0 min/10 min (Arg-0/Lys-H4, Arg-10/Lys-8) and 1 min/20 min (Arg-0/Lys-H4, Arg-10/Lys-8) treated samples, respectively. Mixed lysates were precleared with protein A beads for 1h to reduce nonspecific binding. The two sets of lysates were immnuoprecipitated for 2h with an agarose-conjugated anti-phosphotyrosine antibody (4G10) followed by addition of agarose-conjugated anti-phosphotyrosine P-Tyr 100 for an additional 4h at 4°C. The precipitated complexes were then washed three times with 3 ml of modified RIPA buffer, three times with 3 ml of 50 mM Tris (pH 7). Elution was performed with 5 ml of 50 mM glycine pH 2.4 for 500-ml beads. The eluate was neutralized with a neutralizing buffer [2 M Tris (pH 8.0), 1.5 M NaCl, and 1 mM EDTA, 35 ml per 1 ml of elution buffer] and was concentrated on Centricon Spin columns (3-kDa molecular mass cut off; Millipore). The concentrated eluates were resolved by one-dimensional gel electrophoresis on a NuPAGE 4-12% Bis-Tris gel (Invitrogen).

For Western blotting, differentiated adipocytes were cultured in serum-free medium for 16 h before stimulation with insulin for 1, 5, 10, or 20 min, respectively. The cells were harvested in modified RIPA buffer, and equal amounts of cleared lysates incubated with a mixture of agarose-conjugated 4G10 and P-Tyr-100 antibodies for 6h at 4°C. After three washes with lysis buffer, immunoprecipitated proteins were separated on SDS-PAGE, blotted to a nitrocellulose membrane, and detected with an ECL kit (Amersham Pharmacia).

Peptide Pull-Down, Using Quantitative Mass Spectrometry. Peptides were synthesized by using the Fmoc strategy as described in ref. 5 (Intavis). Peptides were immobilized on Dynabeads MyOne Streptavidin C1 (Dynal) and subsequently incubated with 350 mg of cell extract (diluted of 0.6 mg/ml) in binding buffer [50 mM Tris×HCl (pH8.0), 150 mM NaCl, 0.1% Nonidet P-40, 1 mM DTT, and protease inhibitors] for 2 h at 4°C in a rotation wheel. Beads were washed 5 times with 1 ml of binding buffer containing 400 mM NaCl and finally twice with 1 ml of binding buffer. Beads from both pull-downs (unmodified and modified peptide) were subsequently pooled and bound proteins were eluted and subsequently subjected to in solution digest and MS analysis.

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