In Vivo Profiling Endogenous Interactions with Knock-Out in

Jan 21, 2009 - termed in vivo Profiling Endogenous Interactions with. Knock-out (iPEIK). In our design, mouse embryonic fibroblasts (MEFs) derived fro...
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In Vivo Profiling Endogenous Interactions with Knock-Out in Mammalian Cells Ling Xie,† Linhong Jing,‡ Yanbao Yu,†,§ Kazuhiro Nakamura,| Carol E. Parker,‡ Gary L. Johnson,| and Xian Chen*,†,‡ Department of Biochemistry & Biophysics and Department of Pharmacology, School of Medicine, University of North Carolina, 120 Mason Farm Road, Genetic Medicine, Ste 3010, Campus Box No. 7260, Chapel Hill, North Carolina 27599-7260, UNC-Duke Proteomics Center, Chapel Hill, North Carolina 27599-7260, and Institutes for Biomedical Sciences, Fudan University, Shanghai, China 200032 To precisely identify and screen target-specific proteinprotein interactions at the endogenous level, here we introduce a novel quantitative proteomic method we have termed in vivo Profiling Endogenous Interactions with Knock-out (iPEIK). In our design, mouse embryonic fibroblasts (MEFs) derived from target gene knockout (KO) mice can be stable isotope-tagged and serve as a target-free background to “light-up” the target proteinspecific protein complex formed in the corresponding wild-type (WT) cells. In mass spectrometric analysis of the pairs of non-labeled versus heavy isotope-labeled peptide signals derived from WT versus KO cells, respectively, we then quantitatively measured the abundance differences of the proteins in the complex immunoprecipitated (IP) from the target-expressing WT versus targetabsent KO cells, respectively. Those proteins detected with little or no presence in the cells of KO origin were determined as target-specific interacting partners. Further, dynamic interactors could be identified through different IP mixing schemes. Using iPEIK we identified multiple interacting partners both previously known and unknown to be associated with mitogen-activated protein kinase kinase kinase 2 (MEKK2). Because of the availability of a large library of knockout mice models with various target proteins of biological interests our method is generally applicable to screen any endogenous targetspecific PPIs of physiological relevance. In mamalian cells the low-abundance, transient, and dynamic nature of endogenous protein-protein interactions (PPIs) demand for highly accurate and sensitive methods to distinguish target protein-specific interactions. Immunoprecipitation (IP) is a commonly used method to pull down a target protein and its interacting partners. However, the lack of a threshold to distinguish target-specific binding partners in an IP product with nonspecific contaminants often results in a high degree of falsepositive identifications, and this situation can be pronounced with * To whom correspondence should be addressed. E-mail: xian_chen@ med.unc.edu. Fax: (919) 966-2852. † Department of Biochemistry & Biophysics, University of North Carolina. ‡ UNC-Duke Proteomics Center. § Fudan University. | Department of Pharmacology, School of Medicine, University of North Carolina. 10.1021/ac802161d CCC: $40.75  2009 American Chemical Society Published on Web 01/21/2009

antibodies of low specificity. Tandem affinity purification (TAP) was then developed to isolate protein complexes in high purity prior to mass spectrometric (MS) identification.1,2 In general, multiple steps of epitope affinity-based purifications are required to reduce the contaminating proteins bound non-specifically to a bait protein. As a tradeoff, the repetitive affinity washings often lead to the loss of weak or transient interactions of biological relevance. Also, unlike in yeast cells in which it can be engineered to have only the exogenously tagged target expressing, in mammalian cells the untagged endogenous target protein always express which represents the background affecting the specificity of distinguishing target-specific interactors and the yield of the complex pulled-down through the tagged target protein.3 Because of still limited sensitivity and accuracy of currently available abundance-based mass spectrometric (MS) methods in analyzing low-abundance proteins, most of the PPIs were identified in the protein complexes with a target protein expressing at nonphysiologically relevant levels when the identification of endogenous PPIs is highly challenging.4-6 As an effective way to improve signal specificity, stable isotope labeling (SIL) can assist MS for large-scale protein quantification.7-10 Since 1999, we have been developing a quantitative proteomic strategy of amino acidcoded mass tagging (AACT) 8,11 or SILAC as named by another group12 which has provided a high throughput quantitative (1) Zhou, Q.; Lieberman, P. M.; Boyer, T. G.; Berk, A. J. Genes Dev. 1992, 6, 1964–1974. (2) Zhou, Q.; Lieberman, P. M.; Boyer, T. G.; Berk, A. J. Genes Dev. 1992, 6, 1964–1974. (3) Rigaut, G.; Shevchenko, A.; Rutz, B.; Wilm, M.; Mann, M.; Seraphin, B. Nat. Biotechnol. 1999, 17, 1030–1032. (4) Domon, B.; Aebersold, R. Science 2006, 312, 212–217. (5) Domon, B.; Alving, K.; He, T.; Ryan, T. E.; Patterson, S. D. Curr. Opin. Mol. Ther. 2002, 4, 577–586. (6) Aebersold, R.; Mann, M. Nature 2003, 422, 198–207. (7) Chen, X. In Methods in Molecular Biology/Methods in Molecular Medicine; Human Press, 2005. (8) Chen, X.; Smith, L. M.; Bradbury, E. M. Anal. Chem. 2000, 72, 1134– 1143. (9) Oda, Y.; Huang, K.; Cross, F. R.; Cowburn, D.; Chait, B. T. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 6591–6596. (10) Goodlett, D. R.; Keller, A.; Watts, J. D.; Newitt, R.; Yi, E. C.; Purvine, S.; Eng, J. K.; von Haller, P.; Aebersold, R.; Kolker, E. Rapid Commun. Mass Spectrom. 2001, 15, 1214–1221. (11) Zhu, H.; Pan, S.; Gu, S.; Bradbury, E. M.; Chen, X. Rapid Commun. Mass Spectrom. 2002, 16, 2115–2123. (12) Ong, S. E.; Blagoev, B.; Kratchmarova, I.; Kristensen, D. B.; Steen, H.; Pandey, A.; Mann, M. Mol. Cell. Proteomics 2002, 1, 376–386.

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solution8,13-20 for any comparative analysis of global changes in disease-related protein expression,13,18,21 post-translational modifications,14 and lately PPIs.15 Particularly, to increase the sensitivity and accuracy in distinguishing specific PPI in IP pull-down complexes, we have previously introduced a “dual-tagging” (epitope and AACT isotope tags) quantitative proteomic method22 that integrates the capabilities of natural complex formation, epitope affinity isolation, and “in-spectra” quantitative markers to distinguish systematically those interacting proteins from a large non-specific binding background.22,23 However, in addition to the tedious procedures involved in establishing the cell lines that stably express the bait/target protein close to endogenous levels, the use of exogenous tagging strategy for PPI screening always causes concerns about the epitope-tagged protein that could be different from its endogenous version both at expression level and the controlling elements at transcription step. Recently a method of quantitative IP combined with knockdown (QUICK) was introduced,24 which uses RNA intereference (RNAi) to knockdown target gene for the background control and SILAC as quantitative markers to measure abundance changes of targetspecific interactors in the complexes immunoprecipitated from a wild-type cell line. Inevitably, the sensitivity and accuracy of this quantitative method in judging target-specific interactors versus non-specific background primarily rely on the efficiency of RNAibased bait knock-down as the leftover bait protein in the control line may increase the background for quantitative analysis. Furthermore, protein expression is not always proportional to the amount of RNA, especially for those proteins with long turnover rate. Moreover, when either epitope tagging or QUICK approach to pull down bait-specific immunoprecipitates in mammalian cells are used, the interference and competition for binding from untagged/remaining endogenous counterpart could be a major concern to be addressed. To precisely characterize PPIs occurring in real time in the cell lines derived directly from tissue cells or even later in primary cells, here we report a novel quantitative proteomic method of in vivo Profiling Endogenous Interactions with Knockout (iPEIK). In mass spectrometric analysis, the immunoprecipitates originated from bait knockout mouse cells serve as a “clean” background to distinguish the specific bait(13) Zhu, H.; Pan, S.; Gu, S.; Bradbury, E. M.; Chen, X. Rapid Commun. Mass Spectrom. 2002, 16, 2115–2123. (14) Zhu, H.; Hunter, T. C.; Pan, S.; Yau, P. M.; Bradbury, E. M.; Chen, X. Anal. Chem. 2002, 74, 1687–1694. (15) Gu, S.; Pan, S.; Bradbury, E. M.; Chen, X. Anal. Chem. 2002, 74, 5774– 5785. (16) Chen, X. Methods in Molecular Biology/Methods in Molecular Medicine Book Series; Humana Press: Totowa, NJ, 2004. (17) Gu, S.; Pan, S.; Bradbury, E. M.; Chen, X. J. Am. Soc. Mass Spectrom. 2003, 14, 1–7. (18) Pan, S.; Gu, S.; Bradbury, E. M.; Chen, X. Anal. Chem. 2003, 75, 1316– 1324. (19) Hunter, T. C.; Yang, L.; Zhu, H.; Majidi, V.; Bradbury, E. M.; Chen, X. Anal. Chem. 2001, 73, 4891–4902. (20) Harris, M. N.; Ozpolat, B.; Abdi, F.; Gu, S.; Legler, A.; Mawuenyega, K. G.; Tirado-Gomez, M.; Lopez-Berestein, G.; Chen, X. Blood 2004, 104, 1314– 1323. (21) Gu, S.; Chen, J.; Dobos, K. M.; Bradbury, E. M.; Belisle, J. T.; Chen, X. Mol. Cell. Proteomics 2003, 2, 1284–1296. (22) Wang, T.; Gu, S.; Ronni, T.; Du, Y. C.; Chen, X. J. Proteome Res. 2005, 4, 941–949. (23) Du, Y. C.; Gu, S.; Zhou, J.; Wang, T.; Cai, H.; Macinnes, M. A.; Bradbury, E. M.; Chen, X. Mol. Cell. Proteomics 2006, 5, 1033–1044. (24) Selbach, M.; Mann, M. Nat. Methods 2006, 3, 981–983.

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interacting proteins immunoprecipated from those of wild-type mice. Mitogen-activated protein kinases (MAPKs) are ubiquitously expressed and regulate a wide variety of functions in virtually all cell types (see review, ref 25). Dysregulated MAPK activity is associated with a variety of pathological states, including those arising from inflammation, such as arthritis and inflammatory bowel disease,26,27 as well as the syndromes that include the uncontrolled cellular proliferation and tissue remodeling characteristic of cancer.28 However, little is known about the molecular mechanisms underlying the MAPK-related disease pathogenesis. In this regard, the identification of key components and their interactions in MAPK pathways is essential to understand novel functions of this superkinase family. It is known that MAPKs are the terminal kinase in a three kinase phosphor-relay module, in which MAPKs are phosphorylated and activated by mitogen-activated protein kinase kinase MKKs, which themselves are phosphorylated and activated by mitogen-activated kinase kinase kinases (MKKKs) (see review, ref 29). MEKK2 is one of more than 20 MKKKs known so far. MEKK2 is involved in both extracellular-related kinase 5 (ERK5) and c-Jun N-terminal kinase JNK signal pathways as its binding to different partners such as MEK5 and MKK7 can coordinately control ERK5 and c-Jun Nterminal kinase activation.30 Phosphorylated MEKK2 is regulated by Smurf1, a HECT domain ubiquitin ligase, which controls osteoblast activity and bone homeostasis by targeting MEKK2 for degradation.31 As the first step to systematically reveal MEKK2-specific functional network, as well as to evaluate the effectiveness of our design, we chose to perform an unbiased screening on identifying the interacting partners of MEKK2 at the endogenous level. As MEKK2 knockout mice is vital and fertile, suggesting that it is developmentally dispensable,32 the mouse MEKK2-/- and wild-type MEF cell lines which are derived from the embryo of MEKK2 knockout and wild type mice, respectively, 32 are used in our study. EXPERIMENTAL METHODS Cell Culture and Reagents. Wild-type and MEKK2-/- mouse embryonic fibroblasts (MEFs) were isolated as described previously and grown in Dulbecco’s Modification of Eagle’s Medium with 10% fetal bovine serum, 100 U/mL penicillin, and 100 µg/mL streptomycin at 37 °C with 5% CO2.32 For a SILAC/ AACT experiment, MEKK2-/- MEF cells were grown in the regular leucine-depleted DMEM later supplemented with 110 mg/L of D3-L-Leucine (Cambridge Isotope Laboratories) while wild-type MEF cells were cultured in the regular medium. A mouse monoclonal antibody (mAb) for MEKK2 was generated against recombinant MEKK2. Immunoprecipitation and Immunoblotting Analysis. The MEF cells were washed with ice-cold phosphate-buffered saline (25) Pearson, G.; Robinson, F.; Beers Gibson, T.; Xu, B. E.; Karandikar, M.; Berman, K.; Cobb, M. H. Endocr. Rev. 2001, 22, 153–183. (26) Johnson, G. L.; Lapadat, R. Science 2002, 298, 1911–1912. (27) Hollenbach, E.; Neumann, M.; Vieth, M.; Roessner, A.; Malfertheiner, P.; Naumann, M. FASEB J. 2004, 18, 1550–1552. (28) Gollob, J. A.; Wilhelm, S.; Carter, C.; Kelley, S. L. Semin. Oncol. 2006, 33, 392–406. (29) Cuevas, B. D.; Abell, A. N.; Johnson, G. L. Oncogene 2007, 26, 3159–3171. (30) Nakamura, K.; Johnson, G. L. Mol. Cell. Biol. 2007, 27, 4566–4577. (31) Yamashita, M.; Ying, S. X.; Zhang, G. M.; Li, C.; Cheng, S. Y.; Deng, C. X.; Zhang, Y. E. Cell 2005, 121, 101–113. (32) Garrington, T. P.; Ishizuka, T.; Papst, P. J.; Chayama, K.; Webb, S.; Yujiri, T.; Sun, W.; Sather, S.; Russell, D. M.; Gibson, S. B.; Keller, G.; Gelfand, E. W.; Johnson, G. L. EMBO J. 2000, 19, 5387–5395.

and lysed with solubilizing buffer (1% NP-40, 10 mM Tris [pH 7.5], 150 mM NaCl, 0.4 mM EDTA, 2 mM Na3VO4, 1× phosphotase inhibitor cocktail (Pierce), 1× protease inhibitor cocktail (sigma-aldrich)) on ice. The cell lysate was centrifuged at 100,000g for 60 min, and supernatants were retained for further processing. For IP, the monoclonal anti-MEKK2 was first coupled to protein G agarose (Genscript) with dimethyl pimelimidate. For a post-mixing IP experiment, 20 µg of conjugated antibody was incubated with the lysate from approximately 1-2 × 108 MEKK2-/- and wild-type MEF cells, separately, for 6 h at 4 °C. The immunoprecipitates were collected by centrifugation followed by washing with solubilization buffer three times and PBS twice. The immunoprecipitated complex was eluted with 0.1 M Glycine (pH2.5). The protein concentration was determined by BCA assay. The eluted proteins from WT and KO were mixed by 1:1 and precipitated with 1/100 (v/v) of 2% Na deoxycholate and 1/10 of 100% Trichloroacetic acid overnight at 4 °C. The precipitates were redissolved in 1× SDS gel sample buffer at 70 °C and separated on NuPAGE 4-12% SDS gel (Invitrogen). The gel was stained by G-250 and contiguously cut for in-gel digestion. For a premixing IP experiment, the WT and KO cell lysates were mixed by the same protein mass followed by immunopreciptation as described above. For immunobloting, 5 mg of cell lysate was incubated with 1 µg of anti-MEKK2 tethered on protein G agarose. The immune complexes were washed, eluted, separated by SDS-PAGE, and transferred to a PVDF membrane. After blocking, membranes were blotted with selected antibodies and visualized using the ECL plus detection system (GE). In-Gel Digestion and Reverse-Phase NanoLC-MS/MS Analysis. The gel bands were loaded on ProGest autodigester (Genomic Solutions) for tryptic digestion. The extracted peptide solution was dried with speed-vac. The peptide pellets were redissolved in 0.5% acidic acid, desalted with reversed phase C18-packed tips, and eluted with 80% acetonitrile/0.5% acetic acid. The organic solvent was removed by speed-vac. The volume of digested peptides was brought up in 0.1% formic acid. Each sample was separated with online Eksigent nanoLC system and analyzed by a LTQ-Orbitrap hybrid mass spectrometer (Thermo Electron, San Jose, CA), which was equipped with a nano electrospray source (New Objective, Inc., Woburn, MA). The peptides were loaded on IntegraFrit Sample trap (ProteoPep II C18, 300 Å, 5 µm, 75 µm × 25 mm, New Objective, Inc., Woburn, MA) by using mobile phase of 50% acetonitrile in 0.1% formic acid. The retained peptides were washed isocratically with the same buffer to remove any excess reagents. The cleaned peptides were resolved on a PicoFrit Analytical Columns (ProteoPep II C18, 300 Å, 5 µm, 50 µm × 100 mm, tip ID ) 10 µm, New Objective, Inc., Woburn, MA) or Dionex 75 um × 150 mm column with a multistep gradient of solvent 2A (water premixed with 0.1% formic acid) and solvent 2B (acetonitrile premixed with 0.1% formic acid). Data Analysis. The LC-MS-MS/MS raw data was converted to DTA files using ThermoElectron Bioworks 3.3.1 and correlated to theoretical fragmentation patterns of tryptic peptide sequences from the Fasta databases using SEQUEST. Search parameters included (1) variable modifications allowing mass increase of 80

Da for possible phophorylation and 3 Da for 3-deuterium labeled Leucine; (2) restricted to trypsin generated peptides, allowing for two missed cleavages; (3) The criteria for peptide were based on top hit(s) with individual cross correlation exceeding a threshold dependent on the precursor charge state. The proteins matched with at least two peptides and probability smaller than 0.001 (P < 0.001) were considered as positive identification. PepQuan was used in SILAC protein quantification. RESULTS AND DISCUSSION General iPEIK Experimental Design. As illustrated in Figure 1, KO cells are cultured in a “heavy” medium supplemented with particular type of heavy amino acids, for example, a selected type of either deuterium, or/ 13C, or/ 15N-enriched amino acids while WT cells are grown in the “light/regular” medium. Each WT or KO cell lysate is incubated with monoclonal anti-MEKK2 covalently coupled to the protein G/A-agarose or magnetic beads for IP. The heavy and light immunoprecipitates are then combined at a 1:1 ratio of total protein mass in a “post-IP mixing” scheme (Figure 1 right). Also to further distinguish those specific interactors with high on- and off-rate in their binding to the target protein, we also perform a single IP experiment on the mixed WT and KO cell lysates in a “pre-IP mixing” scheme (Figure 1 left). The proteins in each set of immunoprecipitates are separated on 1D-SDS gels, digested, and the eluted peptides from each gel slice are subjected to LC-MS/MS analysis. In MS spectra, the peptide signals from target/bait protein should show only the light isotope peak. Its specific binding proteins should present high light-to-heavy isotope ratio (L/H) while non-specific contaminants show equal intensity for both light and heavy isotope signals. Moreover, if some interactors are detectable in both post-IP and pre-IP mixing scheme, the dynamic interacting components can display different L/H ratios for their peptide signals, depending on the approach taken through either post-IP or pre-IP mixing scheme. Because of possible back-exchanges between light and heavy version of a protein during the IP incubation step, we expect to observe a relatively lower L/H for a target-specific interactor in the pre-IP mixing than that in the post-IP mixing. Profiling the Immunoprecipitated Complex Associated with MEKK2. As shown in Figure 2, in both pre-IP and post-IP pair mixing runs, the bait MEKK2 was identified with no signal at the m/z corresponding to its heavy leucine-containing peptides, indicating the absence of the bait protein from MEKK2-/- cells (Figure 2a). Multiple peptides of MEKK2 representing approximately 40% of its full-length sequence coverage were identified with similar infinite L/H ratio. Using either post-IP or pre-IP mixing approach, we identified 96 and 130 proteins respectively with at least two peptides sequenced for each protein identification at high confidence (p < 0.001) (see Supporting Information, Tables 1 and 2). Among them, 86 and 114 proteins had multiple leucinecontaining peptides for quantification of L/H ratios, respectively. The peptide signals from most of those proteins such as Actin showing their L/H ratio around 1 (Figure 2b) were considered as non-specific contaminants because they were randomly and equally associated with the antibody beads regardless of the presence or absence of MEKK2 in the cells. In reference to the measured L/H ratio of certain proteins previously known to interact with MEKK2, we set the threshold for Analytical Chemistry, Vol. 81, No. 4, February 15, 2009

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Figure 1. Schematic diagram of iPEIK design for screening endogenous PPIs. Both MEF cell lines with either the target gene knockout or the wild-type with endogenous gene retained can be generated from particular tissue or organ of the corresponding knockout and wild-type mice, respectively. When a selected MEF cell line is cultured in the media containing a particular type of stable isotope-enriched or heavy amino acids, AACT can be metabolically incorporated into the cellular proteins in a residue-specific way. Depending upon the purpose of learning the nature of the interactions, either post-IP mixing or pre-IP mixing scheme can be separately used or both used for comparative analysis. The “bait” protein (indicated by blue oval), its direct binding partners (triangle/stable, moon/dynamic) and indirect interacting protein (rectangular) are pulled down along with some non-specific contaminants (diamond). Color: blue coded proteins are from wild-type cells in light media, red ones from knockout cells in heavy media.

determining new MEKK2-interacting proteins identified by MS as follows: an L/H ratio over 2 or a 100% increase in abundance by comparing the peptides extracted from wild-type versus KO cells through post-IP mixing scheme, while following pre-IP mixing 1414

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scheme, the L/H ratio was set at over 1.3 because of possible backexchange effect. The proteins with relatively high L/H ratios were listed in Table 1. The distribution of L/H ratios versus the number of the identified proteins was given in Figure 2e.

Figure 2. Identification of endogenous interacting partners in the immnuoprecipitates. (a) Representative MS spectra for MEKK2 peptides showed that MEKK2 is only present in light form from wild-type cells and there is no signal originated from knockout cells. (b) The majority of protein components, such as Actin, in the complex were identified with no abundance change between their light and heavy forms; (c, d) MEK5 and 14-3-3 are known interacting proteins with MEKK2. (e) The open circles are the L/H ratios of proteins identified following post-IP mixing scheme, and the filled circles correspond to those following pre-IP mixing scheme. The details of all identified proteins are listed in Supporting Information, Tables 1 and 2. (f) Some proteins, such as Heat Shock protein 90 R, are identified with significantly higher ratio in post-IP scheme than that in pre-IP scheme. This may indicate their dynamic or weak interactions with the bait protein. Analytical Chemistry, Vol. 81, No. 4, February 15, 2009

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Table 1. Summary of Proteins Identified in the MEKK2 Complex no. of peptides accession no.

protein

total

quantification

L/H ratio

IPI00117088.2 IPI00117846.1 IPI00330804.3 IPI00129517.1 IPI00114407.2 IPI00131871.1 IPI00331738.4 IPI00135660.3

Post-IP Mixing Scheme Mitogen-activated protein kinase kinase kinase 2 Death-associated protein kinase 3 Heat shock protein HSP 90-alpha Isoform Mitochondrial of Peroxiredoxin-5, mitochondrial precursor THO complex 4 COP9 signalosome complex subunit 4 52 kDa Ro protein Serum deprivation-response protein pSDPR

14 2 2 3 2 2 5 3 1

10 2 2 1 1 1 5 2 1

∞ ∞ 3.96 3.80 3.56 3.55 2.49 2.08 24.4

IPI00116498.1 IPI00227392.4 IPI00230682.6 IPI00230707.5 IPI00118384.1

14-3-3 14-3-3 14-3-3 14-3-3 14-3-3

4 2 3 3 4

3 2 2 3 3

4.55 3.46 2.15 2.04 1.85

IPI00116281.2 IPI00118678.1 IPI00320217.8 IPI00116277.2 IPI00469268.4

T-complex T-complex T-complex T-complex T-complex

2 2 3 4 5

1 1 2 2 2

3.16 2.70 2.25 2.25 2.08

IPI00117088 IPI00121471 IPI00126447 IPI00177038 IPI00330497 IPI00230435 IPI00400300 IPI00119478

Pre-IP Mixing Scheme Map3k2 Mitogen-activated protein kinase kinase kinase 2 Serpinb6a Map2k5 Isoform 1 of Dual specificity mitogen-activated protein kina Actr2 Actin-related protein 2 Kank2 Isoform 1 of KN motif and ankyrin repeat domain-containing Lmna Isoform C2 of Lamin-A/C Lmna Isoform C of Lamin-A/C Tmod3 Tropomodulin-3

17 2 3 2 3 2 3 3

14 2 1 2 2 1 2 3

∞ 1.64 1.60 1.60 1.51 1.50 1.37 1.30

protein protein protein protein protein

zeta/delta eta beta/alpha gamma epsilon

protein 1 subunit zeta protein 1 subunit alpha A protein1 subunit beta protein 1 sununit delta protein 1 subunit theta

Detection of Known MEKK2-Interacting Proteins in the Immunoprecipitates Derived from WT Cells in Contrast to the KO Background. By using iPEIK approach a few proteins previously known for their involvements in the MAPK signaling pathway were identified as the MEKK2 interactors, which include MEK5, heat shock protein 90R and 14-3-3 proteins (Table 1). Those proteins have been previously found to bind MEKK2 by either two-hybrid screening assay, or tandem affinity purification, or in vitro pull-down assay with over-expressed MEKK2 protein.33-35 In the post-IP mixing complex, the L/H ratios of 3.98 and 4.55 were found for Hsp90aa1 and14-3-3 zeta/delta, respectively, suggesting their specific interactions with MEKK2 (Figure 2d and 2f left). Interestingly, MEK5 was identified in the pre-IP mixing with L/H at 1.6 after averaging all populations of light and the deuterium-containing MEK5 peptide signals possibly offset during the µLC elution. The heavy population of the MEK5 peptides was probably due to fast exchange between “light” MEK5 bound to MEKK2 in wild-type cells and the free “heavy” unbound MEK5 expressed in KO cells when the heavy and light cell lysates were mixed. Indeed, the high on-rate and off-rate of in vitro binding between MEK5 and MEKK2 measured by Biacore indicated these highly dynamic interactions between both proteins (ka ) 5.8 × 105 M-1 s-1, kd ) 0.055 s-1, unpublished data). Another possibility is that free MEKK2 in WT cell lysate interacted with (33) Bouwmeester, T.; Bauch, A.; Ruffner, H.; Angrand, P. O.; Bergamini, G.; Croughton, K.; Cruciat, C.; Eberhard, D.; Gagneur, J.; Ghidelli, S.; Hopf, C.; Huhse, B.; Mangano, R.; Michon, A. M.; Schirle, M.; Schlegl, J.; Schwab, M.; Stein, M. A.; Bauer, A.; Casari, G.; Drewes, G.; Gavin, A. C.; Jackson, D. B.; Joberty, G.; Neubauer, G.; Rick, J.; Kuster, B.; Superti-Furga, G. Nat. Cell Biol. 2004, 6, 97–105. (34) Fanger, G. R.; Widmann, C.; Porter, A. C.; Sather, S.; Johnson, G. L.; Vaillancourt, R. R. J. Biol. Chem. 1998, 273, 3476–3483. (35) Nakamura, K.; Johnson, G. L. J. Biol. Chem. 2003, 278, 36989–36992.

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free MEK5 in the MEKK2-/- cell lysate when they are mixed for IP. Similarly, the L/H ratio of HSP90aa1 was found close to 1 in the pre-IP mixing complex in comparison with that of 4.55 in the post-IP mixing one (Figure 2f). This may also be true for other proteins identified in the immunoprecipitates following pre-IP mixing scheme, such as TRAF7 and isoform1a of C-jun-amino-terminal kinase-interacting protein 3 (Mapk8ip3) (Supporting Information, Table 2). As shown in Figure 2e, more proteins with high L/H ratios were identified in the post-IP scheme than those using the pre-IP mixing approach. Apparently, pre-IP mixing simplifies the experimental procedure and decreases the abundance difference for non-specific binding proteins from two cell populations, but it could be problematic for those proteins bound to the enzymes through weak and/or dynamic interactions. If an interactor is detected in both approaches, the comparative analysis of its ratios could suggest its binding strength to the target/bait protein. Newly Identified Components in the MEKK2-Interacting Complex. In addition to these known interactors, the proteins identified with L/H ratios larger than 2 were considered as possible novel MEKK2-interacting partners. These identifications include Zipper interacting protein kinase (ZIPK, also known as death-associated protein kinase 3 [DAPK3]), Ro 52 protein (Trim21), serum deprivation response protein (SDPR), and so forth. (Figure 3 and Table 1). Similar to the bait MEKK2, ZIPK was identified with an infinite L/H ratio or no signal detected from its heavy version immunoprecipitated from the KO cell line (Figure 3a). Ro52 was also identified with L/H at 2.49 (Figure 3b). Because of its L/H ratio at close to the threshold observed in MS analysis, Ro52 was chosen for further validation by using

Figure 3. Newly identified MEKK2-interacting partners. Possible novel MEKK2-interacting partners are identified with L/H ratio higher than 2, such as Zipper interacting protein kinase (Zipk) (a), Ro52 protein (Trim21) (b), serum deprivation response protein (SDPR) and its phosphorylated form (pSDPR) (c, left and right) (see Supporting Information).

Figure 4. Validation of selected components in MEKK2 immunoprecipitate using immunoblotting. Left panel: The immunoprecipitates obtained from MEKK2-/- or wild type MEF cells by using antiMEKK2 antibody were immunoblotted with the corresponding antibody of each selected protein. Right panel: whole cell lysate (WCL) were immunoblotted with the corresponding antibody of each selected protein.

co-IP and Western blotting. As shown in Figure 4 (left panel), Ro52, as well as MEK5, was detected only in the immunoprecipitates from wild-type MEF along with MEKK2. Meanwhile, the cellular expression of both MEK5 and Ro52 were found unchanged comparing their abundances available in WT versus KO cells (Figure 4 right panel). This observation explains why the residual population of either MEK5 or Ro52 was observed at their heavy isotope signals, that is, because of their expressions also in KO cells, small populations of these proteins may come down during the IP experiments performed in target-absence KO cells. Meanwhile MS is more sensitive to detect these populations. Interestingly among those newly identified interactors, both non- and phospho-peptides belonging to SDPR were identified. The L/H ratio of the SDPR nonphosphopeptides was at 2 when comparing its population in MEKK2 versus MEKK2-/- immu-

noprecipitates (Figure 3c left). Meanwhile a significantly high L/H at approximately 24 was observed for the SDPR phosphopeptide, suggesting that the phosphorylated SDRP is predominantly involved in the MEKK2 complex (Figure 3c right). Further, the validation and characterization of other possible MEKK2-specific components in the identified MEKK2 complex are carried out by using a variety of biological assays, and the corresponding results will be reported else. In conclusion, the success of our method relies on the tagging with stable isotopes at the complexes immunoprecipitated from the cells derived from wild-type or target protein-depleted knockout mice. In MS analysis, the isotopic signals observed for the bait protein derived from knockout mouse cells are serving as a “clean” background from which to distinguish the specific baitinteracting proteins immunoprecipitated from those of wild-type origin. To decode biologically relevant protein interaction networks in vivo, iPEIK is generally applicable to those biological systems with available knockout mice models. ACKNOWLEDGMENT This work was supported by U.S. NIH 1R01AI064806-01A2, and U.S. Department of Energy, the Office of Science (BER), Grant DE-FG02-07ER64422. SUPPORTING INFORMATION AVAILABLE Summary of proteins in MEKK2 complex with pre- and postIP mixing scheme. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review October 12, 2008. Accepted January 6, 2009. AC802161D Analytical Chemistry, Vol. 81, No. 4, February 15, 2009

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