Impact of Detergents on Membrane Protein Complex Isolation

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Impact of detergents on membrane protein complex isolation Yu-Chen Lee, Jenny Arnling Bååth, Ryan M. Bastle, Sonali Bhattacharjee, Mary Jo Cantoria, Mark Dornan, Enrique Gamero-Estevez, Lenzie Ford, Lenka Halova, Jennifer Kernan, Charlotte Kürten,, Siran Li, Jerahme Martinez, Nalani Sachan, Medoune Sarr, Xiwei Shan, Nandhitha Subramanian, Keith Rivera, Darryl J. Pappin, and Sue-Hwa Lin J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.7b00599 • Publication Date (Web): 07 Nov 2017 Downloaded from http://pubs.acs.org on November 9, 2017

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Journal of Proteome Research

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Impact of detergents on membrane protein complex isolation Yu-Chen Lee†, Jenny Arnling Bååth‡, Ryan M. Bastle‡, Sonali Bhattacharjee‡, Mary Jo Cantoria‡, Mark Dornan‡, Enrique Gamero-Estevez‡, Lenzie Ford‡, Lenka Halova‡, Jennifer Kernan‡, Charlotte Kürten‡, Siran Li‡, Jerahme Martinez‡, Nalani Sachan‡, Medoune Sarr‡, Xiwei Shan‡, Nandhitha Subramanian‡, Keith Rivera‡, Darryl Pappin‡, Sue-Hwa Lin*† †Departments of Translational Molecular Pathology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030, United States ‡ Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724, United States This work was performed during the Cold Spring Harbor Laboratory “Protein Purification and Characterization” course, March 29-April 11, 2017. The authors were course participants and contributed equally to the work.

Key words: membrane protein complex, detergents, cadherin-11, iTRAQ, LC-MS/MS Correspondence: Sue-Hwa Lin: Fax: 713-834-6084; E-mail: [email protected].

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Abstract Detergents play an essential role during the isolation of membrane protein complexes. Inappropriate use of detergents may affect the native fold of the membrane proteins, their binding to antibodies, or their interaction with partner proteins. Here we used cadherin11 (Cad11) as an example to examine the impact of detergents on membrane protein complex isolation. We found that mAb 1A5 could immunoprecipitate Cad11 when membranes were solubilized by dodecyl maltoside (DDM) but not by octylglucoside, suggesting that octylglucoside interferes with Cad11-mAb 1A5 interaction. Further, we compared the effects of Brij 35, Triton X-100, cholate, CHAPSO, zwittergen 3-12, deoxyBIG CHAP, and digitonin on Cad11 solubilization and immunoprecipitation. We found that all detergents, except Brij35, could solubilize Cad11 from the membrane. Upon immunoprecipitation, we found that β-catenin, a known cadherin-interacting protein, was present in Cad11 immune complex among the detergents tested except Brij 35. However, the association of p120 catenin with Cad11 varied depending on the detergents used. Using isobaric tag for relative and absolute quantitation (iTRAQ) to determine the relative levels of proteins in Cad11 immune complexes, we found that DDM and Triton X-100 were more efficient than cholate in solubilization and immunoprecipitation of Cad11 and resulted in the identification of both canonical and new candidate Cad11-interacting proteins.


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Introduction Membrane proteins play a role in a vast array of cellular activities including signaling, ion transport, and adhesion (1). Proteins that interact with membrane proteins are critical in the regulation of cellular signaling and the function of membrane proteins. Isolation and identification of proteins associated with transmembrane proteins or membrane receptors using affinity-purification coupled with mass spectrometry has become an important approach for studying cell signaling (2). Unlike soluble proteins, identification of proteins that interact with membrane proteins requires the use of detergents to solubilize the complex from the membranes. Membrane proteins are susceptible to unfolding and aggregation when solubilized in detergents (3). This may result in alteration of their native structure and a disruption in protein-protein interactions and biological function(s) (4). In this study we employed cadherin-11 (Cad11), a cadherin family adhesion molecule, to investigate the effects of detergents on Cad11 protein complex composition. Cad11 is a calcium-dependent adhesion molecule mainly expressed in mesenchymal cells, including osteoblasts (5). Aberrant expression of Cad11 in prostate or breast cancer cells has been shown to increase their migration in vitro (6) and metastasis to bone in vivo (7-9). Cadherin family proteins, including E-cadherin and N-cadherin, are known to interact with canonical cadherin-interacting proteins, i.e., β-catenin, γ-catenin (junctional plakoglobin), and p120-catenin (δ-catenin). Similar to other members of cadherin family proteins, Cad11 contains a juxtamembrane domain and a catenin-binding domain that interacts with the canonical cadherin-binding proteins (10-12). However, Cad11 likely interacts with additional proteins to perform its unique cellular functions. Herein, we examined the effect of several commonly used detergents on Cad11 protein complex solubilization and co-immunoprecipitation. We solubilized Cad11 in various detergents and found that detergent selection impacted the ability of the antiCad11 antibody to pull down both Cad11 and the associated proteins in the Cad11 complex. Further, we used mass spectrometry to measure and compare the composition



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of the Cad11 protein complex isolated using different detergents. We found that solubilization of Cad11 with select detergents not only preserved association with known interacting proteins but also led to the identification of a new set of candidate Cad11interacting proteins. This work highlights the importance of selecting the appropriate detergent to achieve optimal cellular solubilization and immunoprecipitation of the desired protein complexes. Moreover, our findings suggest that analyses of the composition of co-immunoprecipitated complexes across a panel of detergents can aid in the identification of new candidate interacting proteins.

Materials and Methods Materials PC3-mm2 was a subline derived from PC3 prostate cancer cells (13). Complete mini EDTA free cocktail protease inhibitor was from Roche (Indianapolis, IN). DDM and Big CHAP were from Anatrace (Maumee, OH). CHAPSO was from BioRad (Hercules, CA). Zwittergen 3-12, Brij35, sodium cholate, digitonin, and phenylmethylsulfonyl fluoride were from Sigma Aldrich (St. Louis, MO). mAb 1A5 was generated as described previously (14). Octylglucoside, TritonX-100, Coomassie blue plus protein assay reagent, Protein A/G agarose beads, mAb 5B2H5, HRP-goat anti-mouse antibodies, b-catenin antibodies, and p120-catenin antibodies were from Thermo Fisher Scientific (Waltham, MA). Clathrin heavy chain mAb and HRP-donkey anti-goat antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Detergent solubilization and immunoprecipitation PC3-mm2 cells, in the form of frozen pellets harvested from 15 10-cm culture plates, were thawed on ice and mixed by votexing. Cells were lysed by repeated freeze and thaw process in 1.2 mL of 50 mM HEPES pH 7.4 containing 1 mM phenylmethylsulfonyl fluoride and protease inhibitor (complete mini EDTA free cocktail protease inhibitor). Protein concentration was determined using Coomassie blue plus protein assay reagent using BSA as a standard. The cell lysates (0.2ml) were aliquoted


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into eppendorf tubes. The calculated amount of detergents were added into the tube and mixed with the cell lysates by inverting the tubes several times and then incubated for 30 min at room temperature. The lysate was centrifuged and the supernatants (solubilized lysate) were collected. To immunoprecipitate Cad11, mAb 1A5 (5 µg) was added to the solubilized lysate and the mixture was incubated for one hour at room temperature. The immune complex was isolated by the addition of 50 µl of Protein A/G agarose beads and incubated for either 2 hours at room temperature or overnight at 4°C. The Protein A/G agarose beads were washed three times with PBS. Immune complexes were separated via electrophoresis on a 4-12% Bis-Tris NuPAGE gel (Life Technologies, Carlsbad, CA) and proteins were detected via staining with Gelcode (Thermo Fisher Scientific) or the proteins were transferred onto nitrocellulose membranes. For immunoblotting, primary antibodies were used at 1:1,000 dilutions and HRP goat anti-mouse secondary antibody was at a 1:5,000 dilution. The signal was detected by enhanced chemiluminescence (ECL) (Thermo Fisher Scientific). Mass spectrometry of gel bands from SDS-PAGE Gel bands cut from SDS-PAGEs were washed once in water and three times in 50% acetonitrile with 25 mM ammonium bicarbonate for 15 min each. The gel bands were then dehydrated in 100% acetonitrile and proteins in the gel were digested in 10 µL trypsin (20 ng/µL) (Promega, Madison, WI) plus 10 µL Rapigest (20 µg/µL) (Waters, Milford, MA) and 30 µL 30 mM ammonium bicarbonate at 37°C for 16 hours. After incubation, the digestion solution was transferred into a new tube and gel pieces were extracted twice in 50 µL of 50% acetonitrile with 5% formic acid for 15 min. The combined digestion and extraction solutions were dried in a speed vacuum concentrator, resuspended in 50 µL of 1% formic acid plus 5 mM ammonium acetate, and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) on an Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific) as described in Bilen et al. (15). Data processing of the MS results was conducted as described in Bilen et al. (15). Isobaric tag for relative and absolute quantitation (iTRAQ) of Cad11 immune complex



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6 PC3-mm2 cells (from 15 10-cm culture plates) were resuspended in 1.2 ml of 50

mM HEPES pH7.4 containing 1 mM phenylmethylsulfonyl fluoride and 1 tablet of Complete mini EDTA free protease inhibitor. The cell suspensions were divided into 4 equal portions and solubilized with four different detergents (Brij 35, DDM, sodium cholate, and Triton X-100) at a detergent to protein ratio of 5. After 30 min, the detergent solubilized lysates were collected by centrifugation. Immunoprecipitation with mAb 1A5 was performed as described above. The Protein A/G agarose beads were collected by low speed centrifugation and were washed three times (30 sec each) with water. The immune complexes on Protein A/G agarose beads were reconstituted with 40 mL of 50 mM triethylammonium bicarbonate buffer (TEAB). Protease Max Surfactant was added to a final concentration of 0.1% and tris(2-carboxyethyl)phosphine (TCEP) was added to final concentration of 5 mM. The samples were then heated to 55°C for 20 min and allowed to cool down to room temperature. Methyl methanethiosulfonate (MMTS) was added to the samples to a final concentration of 10 mM and the samples were incubated at room temperature for 20 min to block free sulfhydryl groups. The samples were digested with 2 mg of sequencing grade trypsin (Promega) overnight at 37°C. After digestion, the supernatants were removed from the beads and dried under vacuum. The peptides were reconstituted in 50 mL of 0.5 M TEAB/70% ethanol and labeled with 4-plex iTRAQ reagents for 1 hour at room temperature as described in Ross et al (16). Labeled samples were then acidified to pH 4 using formic acid, combined and concentrated in vacuum until ~10 mL remained. An Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific) equipped with a nano-ion spray source was coupled to an EASY-nLC 1200 system (Thermo Fisher Scientific). The LC system was configured with a self-pack PicoFrit™ 75-µm analytical column with an 8-µm emitter (New Objective, Woburn, MA) packed to 25 cm with ReproSil-Pur C18-AQ, 1.9 µM material. Mobile phase A consisted of 2% acetonitrile; 0.1% formic acid and mobile phase B consisted of 90% acetonitrile; 0.1% formic acid. Peptides were then separated at a flow rate of 200 nL/min using the following steps: 2% B to 6% B over 1 min, 6% B to 30% B over 84 min, 30% B to 60% B over 9 min, 60% B to 90% B over 1 min, held at 90% B for 5 min, 90% B to 50% B over 1 min. The flow rate was then increased to 500 nL/min at 50% B and held for 9 min.


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spectrometer with the application of a distal 2.3 kV spray voltage and a capillary temperature of 300°C. Full-scan mass spectra (Res=60,000; 400-1600 m/z) were followed by MS/MS using the “Top N” method for selection. High-energy collisional dissociation (HCD) was used with the normalized collision energy set to 35 for fragmentation, the isolation width set to 1.2 and a duration of 10 seconds was set for the dynamic exclusion with an exclusion mass width of 10 ppm. We used monoisotopic precursor selection for charge states 2+ and greater, and all data were acquired in profile mode. Database Searching was performed as follows. Peaklist files were generated by Mascot Distiller (Matrix Science). Protein identification and quantification was carried out using Mascot 2.4 (17) against the UniProt human sequence database (92,919 sequences; 36,868,442 residues). Methylthiolation of cysteine and N-terminal and lysine iTRAQ modifications were set as fixed modifications, methionine oxidation and deamidation (NQ) as variable. Trypsin was used as the cleavage enzyme with one missed cleavage allowed. Mass tolerance was set at 30 ppm for intact peptide mass and 0.3 Da for fragment ions. Search results were rescored to give a final 1% FDR using a randomized version of the same Uniprot human database. Protein-level iTRAQ ratios were calculated as intensity weighted, using only peptides with expectation values < 0.05. As this was a protein immunoprecipitation experiment, no global ratio normalization was applied. Protein enrichment was calculated by dividing the true sample protein ratios by the corresponding control sample ratios.

Results Effect of EDTA on Cad11 protein conformation To examine the effect of several commonly used detergents on Cad11 protein complex isolation, we solubilized Cad11 from cultured cells in various detergents and evaluated the ability of an anti-Cad11 antibody to pull down both Cad11 and the associated proteins in the Cad11 complex using iTRAQ mass spectrometry. The



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experimental scheme for the isolation and characterization of Cad11 immune complex from various detergent-solubilized cell lysates is shown in Figure. 1A. PC3-mm2 is one of the few cell lines that are known to express Cad11 (7), which allowed us to use them in our Cad11 immunoprecipitation studies. mAb 1A5, which binds to an epitope in the extracellular domain of human Cad11 (14), was used for immunoprecipitation of Cad11. mAb 5B2H5, which binds to the cytoplasmic domain of human Cad11 (18), was used for immunoblotting of Cad11. Although it is common to digest cells from tissue culture plates by trypsin, we found that Cad11 is largely degraded during this process (Figure 1B). This is likely due to the presence of EDTA in the trypsin solution, as Cad11 is a calcium-dependent cell adhesion molecule and is susceptible to proteolysis in the absence of calcium. In contrast, Cad11 in PC3-mm2 cells was not degraded when collected by scraping the cells from culture plates (Figure 1B). Thus, all following experiments were performed with PC3-mm2 cells collected by scraping the cells from tissue culture plates. EDTA is also commonly included in the immunoprecipitation buffers to inhibit metalloprotease activity. Initial immunoprecipitation studies using an immunoprecipitation buffer (150 mM NaCl, 1 mM EDTA, 1 mM EGTA (pH 8.0), 10 mM Tris-HCl (pH 7.4), 0.2 mM sodium orthovanadate, 0.2 mM PMSF, 1% Triton X100, 0.5% NP-40) as suggested by the manufacturer resulted in a Cad11 protein with a reduced size (data not shown). These observations suggest that Cad11 was partially degraded during the immunoprecipitation. Thus, EDTA was not included in the buffer in the subsequent immunoprecipitation studies. Effect of detergents on solubilization and Cad11 immunoprecipitation by mAb 1A5 We initially examined the effect of DDM (n-Dodecyl-β-D-Maltopyranoside) and octylglucoside on the solubilization and immunoprecipitation of Cad11. PC3-mm2 cells were solubilized with DDM or octylglucoside at a detergent to protein ratio of 5 or 10. Western blot of the detergent-solubilized lysates showed that these two detergents did not affect Cad11 detection by mAb 5B2H5 (Figure 2A). Upon immunoprecipitation with mAb 1A5 we found that mAb 1A5 immunoprecipitated Cad11 from DDM-solubilized lysates, but not from octylglucoside-solubilized lysates, at a detergent to protein ratio of


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either 5 or 10 (Figure 2B). The detergents did not affect antibody binding to Protein A/G agarose beads as shown by Ponceau S stain (Figure 2B). These observations suggest that solubilization of Cad11 by octylglucoside causes a conformational change that interferes with mAb 1A5 binding to Cad11. We further examined the effects of 7 additional detergents on Cad11 solubilization and immunoprecipitation at a detergent to protein ratio of 5. The detergents used were Brij35, sodium cholate, Triton X-100, CHAPSO, Big CHAP, ZWITTERGEN 3-12, and digitonin. DDM was used as a positive control. Western blot of the detergent solubilized lysates showed that these detergents did not have significant effect on Cad11 detection by mAb 5B2H5, except for Brij 35. Immunoblotting with GAPDH antibody showed that GAPDH, a cytosolic protein, is present in very low levels in Brij35-solubilized fraction compared to other detergents (Figure 3A), suggesting that Brij35 may not be able to solubilize PC3-mm2 proteins efficiently or the detergent induces protein precipitation that results in protein loss during the centrifugation. We then examined the effects of the detergents on the solubilization and detection of several known Cad11-interacting proteins, including β-catenin, p120-catenin, and clathrin. As shown in Figure 3A, except with the use of Brij35, β-catenin was found in all of the detergent-solubilized lysates, although the levels of β-catenin varied among different detergents. Similarly, we detected p120-catenin in all of the detergentsolubilized lysates (Figure 3A). These results suggest that these detergents did not have a significant effect on β-catenin and p120-catenin detection on western blot. In contrast, we did not detect clathrin in any of the detergent-solubilized lysates, suggesting that the epitope for the clathrin antibody may be denatured or clathrin was degraded under the conditions used for solubilization. Effect of detergents on the immunoprecipitation of β-catenin and p120-catenin in Cad11 complexes β-catenin and p120-catenin are two proteins known to associate with cadherin family proteins. We examined the effect of detergents on the association of Cad11 with β-catenin in the immune complex. As shown in Figure 3B, β-catenin was found in Cad11 immune complex when solubilized in all the detergents examined, except in



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Brij35. These results suggest that these detergents did not have significant effect on the association of Cad11 with β-catenin. We then examined the presence of p120-catenin in the Cad11 immune complex. As shown in Figure 3B, we detected low levels of p120catenin in immune complexes from Big CHAP- and digitonin-solubilized lysates, very little p120-catenin in immune complex from Triton X-100, and we did not detect p120catenin in immune complexes from DDM-, Brij35-, cholate, CHAPSO-, and Zwittergent 3-12-solubilized lysates (Figure 3B). These results suggest that preservation of the Cad11 and p120-catenin protein-protein interaction is sensitive to detergent selection. Novel Cad11-interacting proteins in DDM solubilized Cad11 immune complex Next, we used mass spectrometry to identify proteins present in the Cad11 immune complex isolated from the DDM-solubilized lysate. The duplicated samples were separated via electrophoresis and protein bands were detected by Coomassie Blue (Gelcode). Similar protein profiles were detected in both samples (Figure 4). In addition to Cad11 and immunoglobulin heavy and light chains, proteins with apparent molecular weights of 180, 80, 65, and 30 kDa were detected (Figure 4). The upper part of the gel (upper lanes) (Figure 4, marked with blue line), containing proteins with apparent molecular masses greater than the heavy chain of IgG (~ 50 kDa), and the lower part of the gel (lower lanes) (Figure 4, marked with red line), containing proteins with apparent molecular masses between heavy and light chain of IgG (23-50 kDa), were cut out and the proteins in the gels were identified by mass spectrometry. LC-MS/MS identified 92 and 100 proteins, using 1% FDR, in the two “upper lanes” (supplemental Table S1A, S1B). Seventy-one proteins were identified in duplicated samples. Among them, we eliminated those proteins commonly present in mass spectrometry analysis, e.g. keratin, heat shock proteins, RNA binding proteins, etc., and proteins with only a single peptide detected by mass spectrometry. This approach resulted in 12 proteins (Table 1A). By taking into account the molecular mass and the relative abundance, as reflected in the MS protein coverage by number of matched peptides and their emPAI values, we determined the protein in band 1 to be clathrin (16 and 9 peptides in duplicate samples), Band 2 to be Cad11 (35 and 35 peptides), Band 3 to be a mixture of β-catenin (33 and 33 peptides) and junction plakoglobin (also known as


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γ-catenin) (23 and 24 peptides), both of which have an apparent molecular mass around 89 kDa. Based on these criteria, Band 4 is likely to be the T-complex protein (12 and 24 peptides detected covering all 8 isoforms), which have molecular masses around 57-60 kDa. Similarly, the analysis of mass spectrometry results from the “lower lanes” identified 111 and 138 proteins (supplemental Table S2A, S2B). 90 proteins were identified in duplicated samples. Eliminating those proteins commonly present in mass spectrometry analysis and proteins with only a single peptide detected by mass spectrometry resulted in 8 proteins (Table 1B). In addition to Cad11 and β-catenin, we found that Flotillins, including FLOT1 and FLOT2, have combined peptides of 40 and 13 in the duplicate samples (Table 1B). Because the apparent molecular mass of Flotillin is 47 kDa, these proteins are likely located close to the IgG heavy chain in the SDS-PAGE. It is likely that Band 6 is Prohibitins (PHB2 and PHB), which have combined peptides of 11 and 15 in the duplicate samples and apparent molecular masses of 30-33 kDa. Because voltage-dependent anion-selective channel proteins (VDAC2) have 6 peptides each in duplicate samples and an apparent molecular mass of 31 kDa (Table 1B), it is also likely that Band 6 is a mixture of Prohibitins and VDACs. Interestingly, we detected clathrin heavy chain and light chain in “upper lanes” and “lower lanes”, respectively, in the mass spectrometry analysis of Cad11 complex in the DDM solubilized cell extract (Table 1A, B and supplemental Table S1, 2), although clathrin heavy chain was not detected by IP/western blot analysis (Figure 3B). Quantification of Cad11 interacting proteins in detergent-solubilized Cad11 immune complex by iTRAQ We further used quantitative mass spectrometry, i.e., isobaric Tags for Relative and Absolute Quantitation (iTRAQ), to compare the composition and relative abundance of proteins in differentially solubilized Cad11 immune complexes. PC3-mm2 cells were solubilized by DDM, sodium cholate, or Triton X-100 and were then immunoprecipitated by mAb 1A5. Solubilization and immunoprecipitation with Brij 35 was performed in parallel and was used as a negative control. iTRAQ analysis identified 415 proteins (1% FDR) in the immune complexes (supplemental Table S3).



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12 We found that mAb 1A5 pulled down Cad11 and the known Cad11-interacting

proteins. For Cad11, 9 significant peptides were identified. The number of significant matches for β-, γ, and p120-catenin (catenin delta 1) are 7, 2, and 2, respectively, and 6 and 2 for clathrin heavy chain and light chain, respectively (Table 2A). Comparing the relative levels of Cad11 in the immune complexes to the control detergent Brij35, DDM and Triton X-100 gave highest ratios of 23.2 and 22.5, respectively, while sodium cholate gave a ratio of 13.3 (Figure 5A). We then analyzed the relative amount of known Cad11interacting proteins in the immune complex. In the DDM solubilized immune complex, β-catenin had the highest ratio followed by γ-catenin, clathrin light chain, clathrin heavy chain, and p120-catenin (Figure 5A), suggesting the relative levels of associated proteins in the immune complex is likely to be Cad11 > β-catenin> γ-catenin> clathrin light chain> clathrin heavy chain> p120-catenin. Similar patterns were observed in immune complexes from cholate or Triton X-100 solubilized lysates (Figure 5A). Interestingly, in the Triton X-100 solubilized lysate, the p120-catenin showed a higher iTRAQ ratio in the Triton X-100 immune complex compared to those in DDM or sodium cholate (Figure 5A). This is consistent with IP/western shown in Figure 3B, in which we detected a low level of p120-catenin in Triton X-100 immune complex but did not detect p120-catenin in DDM or cholate immune complex. Next, we looked for potential new Cad11-associated proteins in the detergentsolubilized Cad11 immune complex. Cad11 is a plasma membrane protein, so it likely interacts with proteins localized in plasma membranes or cytoplasm. We first selected proteins based on their localization by using Ingenuity Pathway Analysis program. This resulted in 33 plasma membrane proteins and 257 cytoplasm proteins. We then eliminated those proteins with less than 2 peptides detected in mass spectrometry, which resulted in 201 proteins. Among them, T-complex protein 1 subunit, Prohibitin, and VDAC2 proteins, which were identified in the SDS-PAGE gel slices by LC-MS/MS, were also detected in the iTRAQ analysis (Figure 5B). The relative levels of these proteins compared to the control detergent Brij35 were between 3-9 fold higher (Figure 5B and Table 2B). We then looked for other new candidate Cad11-interacting proteins. We consider the candidate proteins to be potential Cad11-interacting proteins if (1) the relative levels


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of these proteins compared to that in the control detergent Brij35 were higher than 3, and (2) their ratio profiles in the different detergents are similar to that of Cad11, i.e., high in DDM and Triton X-100 and low in cholate. We found 70 proteins that fit these criteria (Table 3A, B). Among them, we found several proteins that are commonly present in mass spectrometry analysis (19), including 7 proteasome subunit proteins, 3 eukaryotic translation elongation factors, 3 peroxiredoxins, 14 enzymes in metabolic pathways, etc.. We thus separated these 70 proteins into two groups. One group of proteins are potential Cad11-interacting proteins (Table 3A) and another group of proteins are likely to be contaminants (Table 3B). The proteins were listed according to their ratio to Brij35. The potential Cad11-interacting proteins with the relative levels higher than 5 compared to the control detergent Brij35 were graphed in Figure 6. Further analysis is required to confirm that these newly-identified candidate proteins are Cad11-interacting proteins.

Discussion We used Cad11 as an example to show that detergents used to solubilize the transmembrane proteins may affect the proteins associated with the membrane proteins. By using mass spectrometry and iTRAQ, we compared the types and levels of proteins present in Cad11 immune complexes isolated from cells solubilized by different detergents. Our analyses showed that detergents have an effect on both the interactions of Cad11 with its antibody and with its associated proteins. These observations suggest that depending on the detergent used, different associated proteins may be identified. Thus, an evaluation of detergent effects on the various types of interactions during an immunoprecipitation study will be required when studying membrane protein complexes. Additionally, mass spectrometry and iTRAQ evaluation of co-immunoprecipitated complexes across a panel of detergents can assist in identifying new candidate proteins that interact with a membrane protein of interest. Our studies provide a framework for evaluation and selection of detergents for isolation and identification of immune complexes of membrane proteins.



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14 Antibodies are frequently used in immunoprecipitation without knowledge of

their binding site on the specific proteins. Our studies demonstrated that the conformation of the antibody binding sites on the antigen may be altered by detergents used to solubilize the membrane proteins. This alteration may result in failure to immunoprecipitate the protein of interest in some detergent-solubilized lysates. Thus, testing several detergents for their compatibility with immunoprecipitation should be considered. To effectively use a highly sensitive modality like mass spectrometry to identify interacting proteins, it is important to distinguish specific from non-specific interacting proteins through further validation. Although reciprocal immunoprecipitation is commonly used in confirming the interactions between different proteins, this approach is limited by the availability of antibodies that are able to immunoprecipitate the candidate interacting proteins without interfering with protein-protein interactions. Another approach is to examine the ability of these proteins to interact with their respective deletion mutants. We have used such an approach to confirm the binding of angiomotin with Cad11 (11) and these mutants may be used to validate the new interacting proteins identified in this study. Another approach is to use proximity ligation assay (PLA) that detect protein-protein interactions through the proximity of antibodies that bind to these two proteins (20). This method can detect both transient or weak interactions. We have previously used PLA to demonstrate the interactions between Cad11 and clathrin (10). These methods can be used to confirm the interactions between Cad11 and the proteins identified in this study in the future. Different proteins may interact with a specific membrane protein under various cellular conditions to relay the signaling from the cell surface. In our previous studies, we identified angiomotin as a novel Cad11-interacting protein after depleting the common cadherin-interacting proteins, i.e. β-catenin, from the cytosol using the Ecadherin cytoplasmic domain in a GST-pulldown assay (11). Although functional assays demonstrated a role of angiomotin in regulating Cad11-mediated cell migration, the interaction of angiomotin with endogenous Cad11 was only detectable when the lysates were pre-depleted with the cytoplasmic domain of E-cadherin (11). Given that β-catenin and angiomotin binding sites on Cad11 are adjacent to each other, this juxtaposition has


Page 15 of 36

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15

led us to hypothesize that β-catenin and angiomotin may dynamically interact with Cad11 during different cellular conditions. In the present studies, we did not find angiomotin in Cad11 immune complexes in the four detergents solubilized lysates by iTRAQ analysis. It is possible that depletion of canonical cadherin-interacting proteins may be required for the binding of angiomotin with Cad11, although we also cannot exclude the possibility that detergents may affect the binding of angiomotin with Cad11. Identification of protein partners at different cellular states will be of potential interest in future proteomics research development. In our previous study, we identified clathrin as one of the Cad11-interacting proteins using the Cad11 cytoplasmic domain, expressed and purified from E. coli, by a GST-pull down assay (10). Although we demonstrated that Cad11 interacts with clathrin in PC3-mm2 cells by proximity ligation assay (10), we could not show that endogenous clathrin could be immunopurified with Cad11 in an IP/western blot experiment. We reasoned that this result is due to the transient and low affinity interaction of clathrin with Cad11, as clathrin binds transiently with its substrates in clathrin-mediated endocytosis. In the present study, we find that clathrin was not detected in detergent-solubilized lysates by western blot. However, clathrin heavy chain and light chain were identified in Cad11 immune complex by mass spectrometry, albeit with low signals. Thus, it is likely that detergent solubilization interferes with clathrin detection by western blot or that western blot is less sensitive. Different partner proteins may associate with a specific protein in different cell types. For example, Cad11 is a mesenchymal cadherin mainly expressed in osteoblasts and neuronal cells. We found that Cad11 was expressed in prostate and breast cancer cell lines (7, 8), likely due to epithelial-to-mesenchymal transition of tumor cells. While we identified several Cad11-interacting proteins in PC3-mm2 cells, it is likely that Cad11 interacts with different proteins in osteoblasts or neuronal tissues. Because mAb 1A5 was against human Cad11 and was generated in a mouse, mAb 1A5 recognizes an epitope that is unique to human Cad11 sequence (14). While it will be interesting to compare Cad11-interacting proteins in osteoblasts versus tumor cells, the Cad11interacting proteins in mouse osteoblasts are unknown because we isolated the osteoblasts from mouse calvaria.



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Page 16 of 36

16 Isolation of immune complexes of membrane proteins has become an important

approach for studying cell signaling. Detergents are integral in the extraction and purification of membrane proteins. We have shown that detergent selection can impact protein solubilization and protein-protein interactions leading to alterations in the composition of protein complexes purified by immunoprecipitation. We have also demonstrated that coupled with mass spectrometry, the evaluation of protein complex immunoprecipitation in samples using different detergents can assist in the identification of new candidate interacting proteins. Understanding the effect of detergents on various aspects of immune complex isolation will improve the likelihood of identifying proteins associated with membrane proteins.

Acknowledgement We thank the participants of 2016 Cold Spring Harbor Laboratory “Protein Purification and Characterization” course for providing initial observations for this study. Research reported in this publication was supported by the National Cancer Institutes of Health under Award Number R25CA009481. This work was performed with assistance from the CSHL Mass Spectrometry shared resource, which is supported by the Cancer Center Support Grant 5P30CA045508. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Conflict of Interests Disclose The authors declare no competing financial interest.


Page 17 of 36

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17

Table 1. Proteins identified in the Cad11 immune complex. Proteins in Cad11 immune complex from DDM-solubilized PC3-mm2 cells were separated by SDS-PAGE. (A) Mass spectrometry analyses of proteins in SDS-PAGE with apparent molecular masses of >50 kDa from duplicate samples. (B) Mass spectrometry analyses of proteins in SDSPAGE with apparent molecular masses between 50-25 kDa from duplicate samples. A. Mass spectrometry analyses of proteins in SDS-PAGE with apparent molecular masses of >50 kDa. prot_m prot_seq prot_acc

GN

prot_desc

prot_mass prot_score atches uences

prot_cover emPAI

CLH1_HUMAN

CLTC

Clathrin heavy chain 1

191493

410, 240

16.1, 13.2 0.49, 0.25

87911

1615, 1385 80, 77 35, 35

42.7, 45.1 5.72, 5.74

CAD11_HUMAN CDH11 Cadherin-11

19, 11 16, 9

CTNNB CTNB1_HUMAN 1

Catenin beta-1

85442

1792, 1683 73, 69 33, 33

54.8, 56

PLAK_HUMAN

Junction plakoglobin

81693

834, 880

39, 37 23, 24

36.9, 42.8 2.85, 3.09

60306

40, 113

1, 4

1, 4

1.8, 14.2

0.08, 0.37

57452

47, 77

1, 2

1, 2

2.2, 10.8

0.09, 0.18

57888

59, 93

2, 4

2, 4

5.9, 11.5

0.18, 0.39

59633

44, 55

1, 1

1, 1

1.8, 9.2

0.08, 0.08

60495

73, 126

1, 3

1, 3

2, 16

0.08, 0.27

59329

58, 51

2, 3

2, 3

4.4, 17.3

0.17, 0.27

59583

43, 162

2, 5

2, 5

10, 23.7

0.17, 0.49

57988

71, 76

2, 2

2, 2

5.3, 9.6

0.18, 0.18

JUP

5.71, 6.12

T-complex protein 1 subunit TCPA_HUMAN

TCP1

alpha T-complex protein 1 subunit

TCPB_HUMAN

CCT2

TCPD_HUMAN

CCT4

beta T-complex protein 1 subunit delta T-complex protein 1 subunit

TCPE_HUMAN

CCT5

epsilon T-complex protein 1 subunit

TCPG_HUMAN

CCT3

gamma T-complex protein 1 subunit

TCPH_HUMAN

CCT7

TCPQ_HUMAN

CCT8

eta T-complex protein 1 subunit theta T-complex protein 1 subunit

TCPZ_HUMAN

CCT6A zeta



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 36

18

Footnotes: Prot_acc: protein accession number. GN: gene name. Prot_desc: protein description. Prot_mass: protein mass. Prot_cover: Protein cover. emPAI: exponentially modified protein abundance index

B Mass spectrometry analyses of proteins in SDS-PAGE with apparent molecular masses between 50-25 kDa from duplicate samples. prot_acc

GN

prot_desc

CAD11_HUMAN CDH11 Cadherin-11

prot_mass prot_score

matches sequences prot_cover emPAI

87911

346, 354

19, 20

11, 14

19.7, 21.5

2.24, 0.72

CTNNB CTNB1_HUMAN 1

Catenin beta-1

85442

151, 154

6, 8

5, 5

7.3, 8.3

0.69, 0.22

FLOT1_HUMAN FLOT1

Flotillin-1

47326

1117, 101

36, 4

19, 4

53.6, 12.4

3.3, 0.33

FLOT2_HUMAN FLOT2

Flotillin-2

47035

712, 122

34, 10

21, 9

48.1, 25.5

0.65, 0.92

PHB2_HUMAN

PHB2

Prohibitin-2

33276

137, 217

6, 13

6, 10

34.1, 50.8

0.55, 1.76

PHB_HUMAN

PHB

Prohibitin

29786

114, 99

6, 8

5, 5

18.8, 29.8

0.71, 0.76

VDAC2_HUMAN VDAC2 protein 2

31547

117, 216

7, 8

6, 6

27.2, 27.2

0.23, 0.9

CLCA_HUMAN

27060

44, 42

2, 2

2, 1

7.7, 3.6

0.09, 0.13

Voltage-dependent anion-selective channel

CLTA

Clathrin light chain A

Footnotes: Prot_acc: protein accession number. GN: gene name. Prot_desc: protein description. Prot_mass: protein mass. Prot_cover: Protein cover. emPAI: exponentially modified protein abundance index.


Page 19 of 36

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19

Table 2. iTRAQ analyses of proteins in Cad11 immune complexes from Brij35, DDM, cholate, or Triton X-100-solubilized PC3-mm2 cells. (A) Known Cad11-interacting proteins identified by iTRAQ approach. (B) Common candidate Cad11-interacting proteins identified by LC-MS/MS of SDS-PAGE gel slices and by iTRAQ approach. A. Known Cad11-interacting proteins identified by iTRAQ approach.

No. of No. of Matche sequen

Entrez Gene ID Accession

Symbol

Name

Mass

Score s

ces

emPAI Brij35 DDM

Cholate TX100

P55287

CDH11

cadherin 11

93675

434

19

9

0.5

1

23.22

13.28

22.50

B4DGU4

CTNNB1 catenin beta 1 88603

311

17

7

0.47

1

11.07

7.47

11.21

32204

72

5

2

0.3

1

7.57

4.46

7.00

25377

106

2

2

0.39

1

7.38

2.98

6.45

206058 160

8

6

0.13

1

3.01

1.81

3.55

112710 54

4

2

0.08

1

2.06

2.36

6.16

junction C9J826

JUP

plakoglobin clathrin light

P09496-2

CLTA

chain A clathrin heavy

A0A087WVQ6 CLTC

chain catenin delta

C9JZR2

CTNND1 1

Footnotes: emPAI: exponentially modified protein abundance index



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Page 20 of 36

20

B. Common candidate Cad11-interacting proteins identified by LC-MS/MS of SDSPAGE gel slices and by iTRAQ approach.

ID Accession

Entrez Gene Symbol Name

Mass

J3KPX7

PHB2

36101 205 12

8

1.54

1

3.285 1.316

4.576

P17987

TCP1

66214 291 13

6

0.47

1

5.395 3.563

8.891

63652 294 11

7

0.59

1

4.357 2.78

6.419

61222 110 4

4

0.32

1

6.614 4.309

8.987

65381 149 3

2

0.14

1

4.5

2.839

7.454

65635 417 13

7

0.57

1

3.74

2.482

6.121

33643 502 12

3

0.46

1

4.133 1.104

6.801

prohibitin 2

t-complex 1 chaperonin containing TCP1 subunit P50991 CCT4 4 chaperonin containing TCP1 subunit B7ZAR1 CCT5 5 chaperonin containing TCP1 subunit Q99832 CCT7 7 chaperonin containing TCP1 subunit P50990 CCT8 8 voltage dependent anion channel A0A0A0MR02 VDAC2 2

No. of No. of Score Matches sequences emPAI Brij35 DDM

Cholate TX100

Footnotes: emPAI: exponentially modified protein abundance index.


Page 21 of 36

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21

Table 3. Candidate Cad11-interacting proteins identified by iTRAQ approach. (A) Possible new candidate Cad11-interacting proteins. (B) Candidate Cad11-interacting proteins identified by iTRAQ approach that are commonly present in mass spectrometry analyses. A. Possible new candidate Cad11-interacting proteins identified by iTRAQ approach. Entrez Gene ID Accession Symbol Name Mass phosphatidyle thanolamine binding P30086 PEBP1 protein 1 23349 reactive intermediate imine deaminase A P52758 RIDA homolog 15782 F8WDS9 LANCL1 LanC like 1 10285 fascin actin-‐ bundling Q16658 FSCN1 protein 1 58675 sorting nexin O60493 SNX3 20336 3 A0A087X0R sorting nexin 6 SNX12 12 21254 FK506 binding P62942 FKBP1A protein 1A 13240 A0A087X0 acyl-‐CoA ACOT2 thioesterase 2 49001 W7 P27797 CALR calreticulin 54308 vasodilator-‐ stimulated phosphoprotei K7ENL7 VASP n 11746 Q15942 ZYX zyxin 65129 N(alpha)-‐ acetyltransfer ase 50, NatE catalytic A0A087WW J2 NAA50 subunit 11304 P60709 ACTB actin beta 44592 MIA family member 3, ER 23504 Q5JRA6 MIA3 export factor 2 G protein subunit alpha P04899 GNAI2 i2 44460 hydroxysteroi d 17-‐beta HSD17B1 dehydrogenas Q99714 e 10 28635 0

No. of No. of Matche seque Score s nces emPAI Brij35 DDM

Cholate TX100

191

12

3

0.71

1

16.648 3.183 20.354

171 63

5 2

2 1

0.69 0.49

1 1

11.872 4.737 14.785 8.765 3.777 14.968

241

8

3

0.24

1

7.941 1.775 6.797

59

2

2

0.51

1

7.696 2.628 13.458

68

3

2

0.48

1

7.432 2.346 11.748

308

11

3

1.55

1

7.041 2.515 6.727

93 89

3 3

2 2

0.19 0.17

1 1

6.245 2.799 5.621 5.799 2.659 7.099

63 281

2 13

1 4

0.42 0.3

1 1

5.723 1.752 5.346 5.637 1.648 4.703

56 2307

2 109

1 19

0.44 11.84

1 1

5.446 2.126 8.322 5.069 4.561 8.822

76

3

1

0.02

1

4.942 2.844 5.401

196

4

2

0.21

1

4.94

107

6

5

1.08

1

4.921 3.011 8.224


3.511 6.682


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 36

22

P55072 VCP A0A0U1RQF 0 FASN P07737 PFN1 A0A087WT T1 PABPC1 Q9BQ69

MACROD 1

P22307

SCP2

P35579

MYH9

C9JFR7

CYCS

P22314

UBA1

P09669

COX6C

H0YNE9

RAB8B

H0YIZ0

SHMT2

P16144

ITGB4

G5EA31

SEC24C

A0A0G2JLD SSBP1 8 P37802 TAGLN2

O95831

AIFM1

P06576

ATP5B

Q96HC4 A2A274

PDLIM5 ACO2

valosin containing protein 96183 fatty acid 28541 synthase 9 profilin 1 16630 poly(A) binding protein cytoplasmic 1 64407 MACRO domain containing 1 38941 sterol carrier protein 2 65728 myosin heavy 25636 chain 9 5 cytochrome c, 14064 somatic ubiquitin like modifier activating 12555 enzyme 1 6 cytochrome c oxidase subunit 6C 9929 RAB8B, member RAS oncogene family 24448 serine hydroxymethy ltransferase 2 30818 integrin 21140 subunit beta 4 5 SEC24 homolog C, COPII coat complex 11623 component 7 single stranded DNA binding 16597 protein 1 transgelin 2 24250 apoptosis inducing factor mitochondria associated 1 72911 ATP synthase, H+ transporting, mitochondrial F1 complex, beta polypeptide 59983 PDZ and LIM domain 5 69092 aconitase 2 95835

237

9

7

0.36

1

4.734 3.142 7.658

213 398

12 14

7 6

0.11 4.74

1 1

4.522 3.071 7.039 4.445 2.093 5.281

162

3

2

0.14

1

4.376 3.514 4.995

195

5

2

0.24

1

4.292 1.781 5.915

315

17

7

0.79

1

4.059 1.092 6.484

93

4

4

0.07

1

3.998 2.604 4.72

204

9

5

3.34

1

3.989 1.403 5.594

227

7

6

0.22

1

3.772 2.981 4.107

50

2

1

0.51

1

3.716 2.596 4.066

155

8

3

0.67

1

3.713 3.088 6.817

112

4

2

0.31

1

3.678 3.2

83

2

1

0.02

1

3.599 1.776 4.813

150

8

5

0.2

1

3.551 2.88

148 220

7 10

3 6

1.11 2.36

1 1

3.538 2.04 3.16 3.515 1.982 5.376

85

3

2

0.12

1

3.375 2.579 4.004

748

32

10

1.68

1

3.277 2.928 5.488

61 354

2 14

2 6

0.13 0.3

1 1

3.259 2.607 4.604 3.195 1.179 4.425


5.331

5.399

Page 23 of 36

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O15143 K7ERT7 P08574 P05556

23 actin related protein 2/3 complex ARPC1B subunit 1B 44814 vesicle amine VAT1 transport 1 15770 CYC1 cytochrome c1 37560 integrin ITGB1 subunit beta 1 96715

72

2

1

0.1

1

3.136 2.288 4.649

104 118

2 2

1 1

0.3 0.12

1 1

3.116 2.919 4.673 3.036 2.5 4.505

66

2

2

0.09

1

3.003 2.038 4.153


B. Candidate Cad11-interacting proteins that are commonly present in mass spectrometry analyses.

ID Accession Symbol P62937

PPIA

A0A0C4DFU2 SOD2 P30405

PPIF

P00338 P06733

LDHA ENO1

H0YKB3 P29401

SORD TKT

P00505

GOT2

P32119

PRDX2

B4DLR8

NQO1

P25788

PSMA3

A0A0A0MSI0 PRDX1 Q13162

PRDX4

P25786

PSMA1

P40926

MDH2

Q5SZU1

PHGDH

Entrez Gene Name Mass peptidylprolyl isomerase A 20162 superoxide dismutase 2 27040 peptidylprolyl isomerase F 24476 lactate dehydrogenas e A 40844 enolase 1 52759 sorbitol dehydrogenas e 14144 transketolase 74031 glutamic-‐ oxaloacetic transaminase 2 51810 peroxiredoxin 2 24040 NAD(P)H quinone dehydrogenas e 1 25805 proteasome subunit alpha 3 31153 peroxiredoxin 1 20981 peroxiredoxin 4 32538 proteasome subunit alpha 1 31410 malate dehydrogenas e 2 39371 phosphoglycer ate 56366

No. of No. of Matche sequen Score s ces emPAI Brij35 DDM

Cholate TX100

760

35

10

8.7

1

14.694 3.844 15.699

84

2

1

0.17

1

13.908 4.767 16.823

566

30

7

2.3

1

11.744 4.493 14.961

532 1544

32 74

7 18

1.06 3.58

1 1

11.59 2.851 15.167 10.693 2.994 10.199

61 1672

3 84

2 24

0.8 3.68

1 1

10.426 3.389 15.035 9.938 3.34 14.285

677

28

7

1.08

1

9.035

4.109 13.715

509

33

9

3.78

1

8.062

1.127 10.449

73

4

2

0.38

1

8.017

2.123 9.389

185

5

2

0.31

1

7.761

3.184 10.482

1147

68

12

12.3

1

7.057

1.087 9.127

184

9

4

0.68

1

5.958

1.134 8.174

222

7

3

0.49

1

5.871

2.658 8.098

718

30

9

2.24

1

5.417

3.917 7.492

213

6

2

0.16

1

5.104

3.875 6.828



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Page 24 of 36

24

dehydrogenas e proteasome P28070 PSMB4 subunit beta 4 30050 71 complement C1q binding I3L3B0 C1QBP protein 21707 118 pyruvate P14618 PKM kinase, muscle 63376 1341 proteasome subunit alpha G3V5Z7 PSMA6 6 31011 216 lactate dehydrogenas P07195 LDHB e B 40506 304 proteasome 26S subunit, Q13200 PSMD2 non-‐ATPase 2 107629 81 proteasome P28074 PSMB5 subunit beta 5 29759 243 eukaryotic translation elongation A0A087X1X7 EEF1D factor 1 delta 74428 212 protein disulfide isomerase family A Q15084 PDIA6 member 6 52414 104 prolyl 4-‐ hydroxylase subunit beta 63998 177 P07237 P4HB eukaryotic translation elongation factor 1 P26641 EEF1G gamma 54843 201 eukaryotic translation elongation P24534 EEF1B2 factor 1 beta 2 28063 258 glycyl-‐tRNA P41250 GARS synthetase 90750 115 proteasome 26S subunit, non-‐ATPase O00231 PSMD11 11 52622 55

3

2

0.32

1

4.94

2.688 8.163

4

1

0.47

1

4.934

2.402 5.586

68

21

3.96

1

4.814

1.759 8.086

6

4

0.72

1

4.58

2.148 7.169

13

4

0.52

1

4.522

2.277 4.05

4

2

0.08

1

4.482

3.176 6.222

6

3

0.53

1

4.442

3.412 6.855

4

3

0.19

1

4.273

3.088 6.434

3

2

0.17

1

3.912

2.649 4.578

4

3

0.22

1

3.726

2.24

7

3

0.26

1

3.689

2.688 5.502

10

3

0.57

1

3.305

1.857 4.1

3

2

0.1

1

3.12

2.445 4.069

2

2

0.17

1

3.036

2.269 5.642


Figure legend


5.135

Page 25 of 36

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25

Figure 1. Effect of trypsin/EDTA digestion on Cad11 protein integrity. (A) Experimental scheme for the isolation of Cad11 immune complex from various detergent-solubilized cell lysates and the analysis of the proteins in the immune complex. (B) Western blot analysis of Cad11 in PC3-mm2 cells collected by trypsinization or by scraping off the plates. Various amount of the collected lysates were loaded onto SDS-PAGE. Cad11 was largely degraded in the samples collected by trypsin/EDTA digestion but remained intact in the samples collected by scraping off the plates. This experiment was repeated 3 times. Figure 2. Effect of detergents on Cad11 immunoprecipitation by mAb 1A5. PC3-mm2 cells were solubilized with DDM or octylglucoside using a detergent to protein (D/P) ratio of 5 or 10. (A) Western blot showed Cad11 was solubilized with DDM and octylglucoside using a D/P ratio of 5 or 10. Ponceau S stain was used as loading control. (B) mAb 1A5 immunoprecipitated Cad11 from DDM solubilized lysate but not from octylglucoside solubilized lysate. The detergents did not affect antibody binding to Protein A/G agarose beads as shown by Ponceau S stain. This experiment was repeated 4 times. Figure 3. Effect of detergents on Cad11 immune complexes. PC3-mm2 cells were solubilized with detergents as indicated using a detergent to protein ratio of 5. (A) Western blot of detergent-solubilized lysates for Cad11, β-catenin, p120-catenin, clathrin, and GAPDH. Cad11, β-catenin, p120-catenin were solubilized by all detergents tested except Brij35. Clathrin was only detected in the total cell lysates but not in detergentsolubilized lysates. GAPDH showed that all detergents, except Brij35, solubilized the cells to about the same degree. (B) Western blot of proteins immunoprecipitated by mAb 1A5. Cad11 and β-catenin were detected in immune complexes isolated from all detergent-solubilized lysates, except Brij35. However, p120-catenin was only detected in immune complexes of Big CHAP and digitonin, but not in DDM, cholate, CHAPSO or Zwittergen 3-12. These results suggest that these detergents may interfere with Cad11 and p120-catenin interaction.



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Figure 4. Mass spectrometry analysis of proteins in Cad11 immune complex isolated from DDM-solubilized PC3-mm2 cells. The samples in duplicate were separated on SDS-PAGE (lanes 1 and 2) and protein bands were detected by Coomassie blue. Gel pieces with molecular weight >50 kDa (marked by blue line) and 50-25 kDa (marked by red line) were cut out and the proteins in the gels were identified by LC-MS/MS. The protein assignments for each band were based on mass spectrometry data. Arrows indicate the heavy and light chain of immunoglobulin. This experiment was repeated 2 times. Figure 5. iTRAQ analyses of proteins in Cad11 immune complexes from DDM, cholate, or Triton X-100 (TX100)-solubilized PC3-mm2 cells relative to the signal detected using the control detergent Brij35. (A) Relative levels of known Cad11-interacting proteins in the Cad11 immune complexes. (B) Relative levels of candidate Cad11-interacting proteins that were also identified by LC-MS/MS of SDS-PAGE gel slices. Figure 6. New candidate Cad11-interacting proteins identified by iTRAQ analyses of proteins in Cad11 immune complexes from DDM, cholate, or Triton X-100 (TX100)solubilized PC3-mm2 cells relative to the signal detected using the control detergent Brij35. Supporting information The following files are available free of charge at ACS website http://pubs.acs.org Supplemental Table S1A. Lane1 upper_human_1%FDR F103824. (xlsx) Supplemental Table S1B. Lane2 upper_human_1%FDR F103826. (xlsx) Supplemental Table S2A. Lane1 lower_human_1%FDR F103827. (xlsx) Supplemental Table S2B. Lane2 lower_human_1%FDR F103825. (xlsx) Supplemental Table S3. iTraq. (xlsx)


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Impact of Detergents on Membrane Protein Complex Isolation

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