Impact of Detergents on Membrane Protein Complex Isolation

Article pubs.acs.org/jpr

Impact of Detergents on Membrane Protein Complex Isolation Yu-Chen Lee,† Jenny Arnling Båat̊ h,‡ 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,‡ and Sue-Hwa Lin*,† †

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Department 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 S Supporting Information *

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 cadherin-11 (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. Furthermore, we compared the effects of Brij-35, Triton X-100, cholate, CHAPSO, Zwittergent 3-12, Deoxy BIG CHAP, and digitonin on Cad11 solubilization and immunoprecipitation. We found that all detergents except Brij-35 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. KEYWORDS: membrane protein complex, detergents, cadherin-11, iTRAQ, LC−MS/MS

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INTRODUCTION

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 vitro6 and metastasis to bone in vivo.7−9 Cadherin family proteins, including E-cadherin and Ncadherin, are known to interact with canonical cadherininteracting proteins, that is, 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

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 affinitypurification 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 © 2017 American Chemical Society

Received: August 23, 2017 Published: November 7, 2017 348

DOI: 10.1021/acs.jproteome.7b00599 J. Proteome Res. 2018, 17, 348−358

Article

Journal of Proteome Research

transferred onto nitrocellulose membranes. For immunoblotting, primary antibodies were used at 1:1000 dilutions, and HRP goat antimouse secondary antibody was at a 1:5000 dilution. The signal was detected by enhanced chemiluminescence (ECL) (Thermo Fisher Scientific).

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 coimmunoprecipitation. We solubilized Cad11 in various detergents and found that detergent selection impacted the ability of the anti-Cad11 antibody to pull down both Cad11 and the associated proteins in the Cad11 complex. Furthermore, we used mass spectrometry to measure and compare the composition 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 Cad11-interacting 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 coimmunoprecipitated complexes across a panel of detergents can aid in the identification of new candidate interacting proteins.

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Mass Spectrometry of Gel Bands from SDS-PAGE

Gel bands cut from SDS-PAGE 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 of trypsin (20 ng/μL) (Promega, Madison, WI) plus 10 μL of Rapigest (20 μg/μL) (Waters, Milford, MA) and 30 μL of 30 mM ammonium bicarbonate at 37 °C for 16 h. After incubation, the digestion solution was transferred to 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

MATERIALS AND METHODS

Materials

Isobaric Tag for Relative and Absolute Quantitation (iTRAQ) of Cad11 Immune Complex

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). Zwittergent 3-12, Brij-35, sodium cholate, digitonin, and phenylmethylsulfonyl fluoride were from SigmaAldrich (St. Louis, MO). mAb 1A5 was generated as previously described.14 Octylglucoside, TritonX-100, Coomassie blue plus protein assay reagent, protein A/G agarose beads, mAb 5B2H5, HRP-goat antimouse antibodies, b-catenin antibodies, and p120-catenin antibodies were from Thermo Fisher Scientific (Waltham, MA). Clathrin heavy chain mAb and HRP-donkey antigoat antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA).

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 four 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 s 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 to room temperature. Methylmethanethiosulfonate (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 h 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 nanoion 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%

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 vortexing. 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 (0.2 mL) were aliquoted into eppendorf tubes. The calculated amount of detergents was added to 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 1 h 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 h 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 349


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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 amounts 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 three times.

acetonitrile and 0.1% formic acid and mobile phase B consisted of 90% acetonitrile and 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, and 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. Eluted peptides were directly electrosprayed into the Fusion Lumos mass spectrometer with the application of a distal 2.3 kV spray voltage and a capillary temperature of 300 °C. Fullscan 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 s 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 2.417 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 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 two times.

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 of the detergents examined, except in Brij-35. These results suggest that these detergents did not have a 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 p120-catenin in immune complexes from Big CHAP- and digitonin-solubilized lysates and very little p120-catenin in immune complex from Triton X-100, and we did not detect p120-catenin in immune complexes from DDM-, Brij-35-, 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.

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 γ-catenin) (23 and 24 peptides), both of which have an apparent molecular mass around 89 kDa. On the basis of these criteria, Band 4 is likely to be the Tcomplex protein (12 and 24 peptides detected covering all 8 isoforms), which has molecular masses around 57−60 kDa. Similarly, the analysis of mass spectrometry results from the “lower lanes” identified 111 and 138 proteins (Supplemental Tables S2A and 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 eight 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 six 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 DDMsolubilized cell extract (Table 1A,B and Supplemental Tables S1 and S2), although clathrin heavy chain was not detected by IP/Western blot analysis (Figure 3B).

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 DDMsolubilized 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 Tables S1A and S1B). Seventy-one proteins were identified in duplicated samples. Among them, we eliminated those proteins commonly present in mass spectrometry analysis, for example, keratin, heat shock proteins, RNA binding proteins, and so on, 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

Quantification of Cad11-Interacting Proteins in Detergent-Solubilized Cad11 Immune Complex by iTRAQ

We further used quantitative mass spectrometry, that is, isobaric Tags for Relative and Absolute Quantitation 352


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Journal of Proteome Research Table 1. Proteins Identified in the Cad11 Immune Complexa,b (A) Mass Spectrometry Analyses of Proteins in SDS-PAGE with Apparent Molecular Masses of >50 kDa prot_acc

GN

CLH1_HUMAN CAD11_HUMAN CTNB1_HUMAN PLAK_HUMAN TCPA_HUMAN TCPB_HUMAN TCPD_HUMAN TCPE_HUMAN

CLTC CDH11 CTNNB1 JUP TCP1 CCT2 CCT4 CCT5

TCPG_HUMAN TCPH_HUMAN TCPQ_HUMAN TCPZ_HUMAN (B) Mass

prot_desc

prot_mass

prot_score

prot_matches

prot_sequences

prot_cover

emPAI

clathrin heavy chain 1 191493 410, 240 19, 11 16, 9 16.1, 13.2 0.49, cadherin-11 87911 1615, 1385 80, 77 35, 35 42.7, 45.1 5.72, catenin beta-1 85442 1792, 1683 73, 69 33, 33 54.8, 56 5.71, junction plakoglobin 81693 834, 880 39, 37 23, 24 36.9, 42.8 2.85, T-complex protein 1 subunit alpha 60306 40, 113 1, 4 1, 4 1.8, 14.2 0.08, T-complex protein 1 subunit beta 57452 47, 77 1, 2 1, 2 2.2, 10.8 0.09, T-complex protein 1 subunit delta 57888 59, 93 2, 4 2, 4 5.9, 11.5 0.18, T-complex protein 1 subunit 59633 44, 55 1, 1 1, 1 1.8, 9.2 0.08, epsilon CCT3 T-complex protein 1 subunit 60495 73, 126 1, 3 1, 3 2, 16 0.08, gamma CCT7 T-complex protein 1 subunit eta 59329 58, 51 2, 3 2, 3 4.4, 17.3 0.17, CCT8 T-complex protein 1 subunit theta 59583 43, 162 2, 5 2, 5 10, 23.7 0.17, CCT6A T-complex protein 1 subunit zeta 57988 71, 76 2, 2 2, 2 5.3, 9.6 0.18, Spectrometry Analyses of Proteins in SDS-PAGE with Apparent Molecular Masses between 50 and 25 kDa from Duplicate Samples

prot_acc

GN

CAD11_HUMAN CTNB1_HUMAN FLOT1_HUMAN FLOT2_HUMAN PHB2_HUMAN PHB_HUMAN VDAC2_HUMAN

CDH11 CTNNB1 FLOT1 FLOT2 PHB2 PHB VDAC2

prot_desc

CLCA_HUMAN

CLTA

cadherin-11 catenin beta-1 flotillin-1 flotillin-2 prohibitin-2 prohibitin voltage-dependent anion-selective channel protein 2 clathrin light chain A

prot_mass

prot_score

matches

sequences

87911 85442 47326 47035 33276 29786 31547

346, 354 151, 154 1117, 101 712, 122 137, 217 114, 99 117, 216

19, 20 6, 8 36, 4 34, 10 6, 13 6, 8 7, 8

11, 14 5, 5 19, 4 21, 9 6, 10 5, 5 6, 6

27060

44, 42

2, 2

2, 1

prot_cover 19.7, 7.3, 53.6, 48.1, 34.1, 18.8, 27.2,

21.5 8.3 12.4 25.5 50.8 29.8 27.2

7.7, 3.6

0.25 5.74 6.12 3.09 0.37 0.18 0.39 0.08 0.27 0.27 0.49 0.18

emPAI 2.24, 0.72 0.69, 0.22 3.3, 0.33 0.65, 0.92 0.55, 1.76 0.71, 0.76 0.23, 0.9 0.09, 0.13

a

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 and 25 kDa from duplicate samples. bProt_acc: protein accession number; GN: gene name; Prot_desc: protein description; Prot_mass: protein mass; Prot_cover: protein cover; and emPAI: exponentially modified protein abundance index.

Table 2. iTRAQ Analyses of Proteins in Cad11 Immune Complexes from Brij-35, DDM, Cholate, or Triton-X-100-Solubilized PC3-mm2 Cellsa,b (A) Known Cad11-Interacting Proteins Identified by iTRAQ Approach ID accession P55287 B4DGU4 C9J826 P09496-2 A0A087WVQ6 C9JZR2 (B) ID accession

symbol

Entrez gene name

mass

score

no. of matches

no. of sequences

emPAI

Brij-35

DDM

cholate

CDH11 cadherin 11 93675 434 19 9 0.5 1 23.22 13.28 CTNNB1 catenin beta 1 88603 311 17 7 0.47 1 11.07 7.47 JUP junction plakoglobin 32204 72 5 2 0.3 1 7.57 4.46 CLTA clathrin light chain A 25377 106 2 2 0.39 1 7.38 2.98 CLTC clathrin heavy chain 206058 160 8 6 0.13 1 3.01 1.81 CTNND1 catenin delta 1 112710 54 4 2 0.08 1 2.06 2.36 Common Candidate Cad11-Interacting Proteins Identified by LC−MS/MS of SDS-PAGE Gel Slices and by iTRAQ Approach symbol

J3KPX7 P17987 P50991

PHB2 TCP1 CCT4

B7ZAR1

CCT5

Q99832

CCT7

P50990

CCT8

A0A0A0MR02

VDAC2

TX100 22.50 11.21 7.00 6.45 3.55 6.16

Entrez gene name

mass

score

no. of matches

no. of sequences

emPAI

Brij35

DDM

cholate

TX100

prohibitin 2 t-complex 1 chaperonin containing TCP1 subunit 4 chaperonin containing TCP1 subunit 5 chaperonin containing TCP1 subunit 7 chaperonin containing TCP1 subunit 8 voltage dependent anion channel 2

36101 66214 63652

205 291 294

12 13 11

8 6 7

1.54 0.47 0.59

1 1 1

3.285 5.395 4.357

1.316 3.563 2.78

4.576 8.891 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

a

(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. bemPAI: exponentially modified protein abundance index.

(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 353


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complex but did not detect p120-catenin in DDM or cholate immune complex. Next, we looked for potential new Cad11-associated proteins in the detergent-solubilized 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 fewer 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 with the control detergent Brij-35 were between three and nine times 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 of these proteins compared with that in the control detergent Brij-35 were higher than 3, and (2) their ratio profiles in the different detergents were similar to that of Cad11, that is, 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, and so on. 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 Brij-35. The potential Cad11-interacting proteins with the relative levels higher than 5 compared with the control detergent Brij-35 were graphed in Figure 6. Further analysis is required to confirm that these newly identified candidate proteins are Cad11-interacting proteins.

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). We found that mAb 1A5 pulled down Cad11 and the known Cad11-interacting proteins. For Cad11, nine significant peptides were identified. The number of significant matches for β-, γ-, and p120-catenin (catenin delta 1) is 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, whereas sodium cholate gave a ratio of 13.3 (Figure 5A). We then analyzed the relative amount of known

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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 the interactions of Cad11 both 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 coimmunoprecipitated 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. Antibodies are frequently used in immunoprecipitation without knowledge of their binding site on the specific proteins. Our studies demonstrated that the conformation of

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 Brij-35. (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.

Cad11-interacting 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 are 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-100solubilized lysates (Figure 5A). Interestingly, in the Triton-X100-solubilized lysate, the p120-catenin showed a higher iTRAQ ratio in the Triton X-100 immune complex compared with 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 354


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Journal of Proteome Research Table 3. Candidate Cad11-Interacting Proteins Identified by iTRAQ Approacha,b (A) Possible New Candidate Cad11-Interacting Proteins Identified by iTRAQ Approach ID accession P30086 P52758 F8WDS9 Q16658 O60493 A0A087X0R6 P62942 A0A087X0W7 P27797 K7ENL7 Q15942 A0A087WWJ2 P60709 Q5JRA6 P04899 Q99714 P55072 A0A0U1RQF0 P07737 A0A087WTT1 Q9BQ69 P22307 P35579 C9JFR7 P22314 P09669 H0YNE9 H0YIZ0 P16144 G5EA31 A0A0G2JLD8 P37802 O95831 P06576 Q96HC4 A2A274 O15143 K7ERT7 P08574 P05556

ID accession P62937 A0A0C4DFU2 P30405 P00338

symbol

Entrez gene name

PEBP1

phosphatidylethanolamine binding protein 1 RIDA reactive intermediate imine deaminase A homologue LANCL1 LanC like 1 FSCN1 fascin actin-bundling protein 1 SNX3 sorting nexin 3 SNX12 sorting nexin 12 FKBP1A FK506 binding protein 1A ACOT2 acyl-CoA thioesterase 2 CALR calreticulin VASP vasodilator-stimulated phosphoprotein ZYX zyxin NAA50 N(alpha)-acetyltransferase 50, NatE catalytic subunit ACTB actin beta MIA3 MIA family member 3, ER export factor GNAI2 G protein subunit alpha i2 HSD17B10 hydroxysteroid 17-beta dehydrogenase 10 VCP valosin containing protein FASN fatty acid synthase PFN1 profilin 1 PABPC1 poly(A) binding protein cytoplasmic 1 MACROD1 MACRO domain containing 1 SCP2 sterol carrier protein 2 MYH9 myosin heavy chain 9 CYCS cytochrome c, somatic UBA1 ubiquitin like modifier activating enzyme 1 COX6C cytochrome c oxidase subunit 6C RAB8B RAB8B, member RAS oncogene family SHMT2 serine hydroxymethyltransferase 2 ITGB4 integrin subunit beta 4 SEC24C SEC24 homologue C, COPII coat complex component SSBP1 single stranded DNA binding protein 1 TAGLN2 transgelin 2 AIFM1 apoptosis inducing factor mitochondria associated 1 ATP5B ATP synthase, H+ transporting, mitochondrial F1 complex, beta polypeptide PDLIM5 PDZ and LIM domain 5 ACO2 aconitase 2 ARPC1B actin related protein 2/3 complex subunit 1B VAT1 vesicle amine transport 1 CYC1 cytochrome c1 ITGB1 integrin subunit beta 1 (B) Candidate Cad11-Interacting Proteins symbol

PPIA SOD2 PPIF LDHA

Entrez gene name peptidylprolyl isomerase A superoxide dismutase 2 peptidylprolyl isomerase F lactate dehydrogenase A

mass

score

no. of matches

no. of sequences

emPAI

Brij35

DDM

cholate

TX100

23349

191

12

3

0.71

1

16.648

3.183

20.354

15782

171

5

2

0.69

1

11.872

4.737

14.785

10285 58675 20336 21254 13240 49001 54308 11746

63 241 59 68 308 93 89 63

2 8 2 3 11 3 3 2

1 3 2 2 3 2 2 1

0.49 0.24 0.51 0.48 1.55 0.19 0.17 0.42

1 1 1 1 1 1 1 1

8.765 7.941 7.696 7.432 7.041 6.245 5.799 5.723

3.777 1.775 2.628 2.346 2.515 2.799 2.659 1.752

14.968 6.797 13.458 11.748 6.727 5.621 7.099 5.346

65129 11304

281 56

13 2

4 1

0.3 0.44

1 1

5.637 5.446

1.648 2.126

4.703 8.322

44592 235042

2307 76

109 3

19 1

11.84 0.02

1 1

5.069 4.942

4.561 2.844

8.822 5.401

44460 28635

196 107

4 6

2 5

0.21 1.08

1 1

4.94 4.921

3.511 3.011

6.682 8.224

96183 285419 16630 64407

237 213 398 162

9 12 14 3

7 7 6 2

0.36 0.11 4.74 0.14

1 1 1 1

4.734 4.522 4.445 4.376

3.142 3.071 2.093 3.514

7.658 7.039 5.281 4.995

38941 65728 256365 14064 125556

195 315 93 204 227

5 17 4 9 7

2 7 4 5 6

0.24 0.79 0.07 3.34 0.22

1 1 1 1 1

4.292 4.059 3.998 3.989 3.772

1.781 1.092 2.604 1.403 2.981

5.915 6.484 4.72 5.594 4.107

9929 24448

50 155

2 8

1 3

0.51 0.67

1 1

3.716 3.713

2.596 3.088

4.066 6.817

30818 211405 116237

112 83 150

4 2 8

2 1 5

0.31 0.02 0.2

1 1 1

3.678 3.599 3.551

3.2 1.776 2.88

5.331 4.813 5.399

16597

148

7

3

1.11

1

3.538

2.04

3.16

24250 72911

220 85

10 3

6 2

2.36 0.12

1 1

3.515 3.375

1.982 2.579

5.376 4.004

59983

748

32

10

1.68

1

3.277

2.928

5.488

69092 95835 44814

61 354 72

2 14 2

2 6 1

0.13 0.3 0.1

1 1 1

3.259 3.195 3.136

2.607 1.179 2.288

4.604 4.425 4.649

15770 104 2 1 0.3 1 3.116 37560 118 2 1 0.12 1 3.036 96715 66 2 2 0.09 1 3.003 That Are Commonly Present in Mass Spectrometry Analyses

2.919 2.5 2.038

4.673 4.505 4.153

mass

score

no. of matches

no. of sequences

emPAI

Brij35

DDM

cholate

TX100

20162 27040 24476 40844

760 84 566 532

35 2 30 32

10 1 7 7

8.7 0.17 2.3 1.06

1 1 1 1

14.694 13.908 11.744 11.59

3.844 4.767 4.493 2.851

15.699 16.823 14.961 15.167

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Journal of Proteome Research Table 3. continued (B) Candidate Cad11-Interacting Proteins That Are Commonly Present in Mass Spectrometry Analyses ID accession

symbol

P06733 H0YKB3 P29401 P00505

ENO1 SORD TKT GOT2

P32119 B4DLR8

PRDX2 NQO1

P25788 A0A0A0MSI0 Q13162 P25786 P40926 Q5SZU1 P28070 I3L3B0 P14618 G3V5Z7 P07195 Q13200

PSMA3 PRDX1 PRDX4 PSMA1 MDH2 PHGDH PSMB4 C1QBP PKM PSMA6 LDHB PSMD2

P28074 A0A087X1X7

PSMB5 EEF1D

Q15084

PDIA6

P07237 P26641

P4HB EEF1G

P24534

EEF1B2

P41250 O00231

GARS PSMD11

Entrez gene name

mass

score

no. of matches

no. of sequences

emPAI

Brij35

DDM

cholate

TX100

enolase 1 sorbitol dehydrogenase transketolase glutamic-oxaloacetic transaminase 2 peroxiredoxin 2 NAD(P)H quinone dehydrogenase 1 proteasome subunit alpha 3 peroxiredoxin 1 peroxiredoxin 4 proteasome subunit alpha 1 malate dehydrogenase 2 phosphoglycerate dehydrogenase proteasome subunit beta 4 complement C1q binding protein pyruvate kinase, muscle proteasome subunit alpha 6 lactate dehydrogenase B proteasome 26S subunit, non-ATPase 2 proteasome subunit beta 5 eukaryotic translation elongation factor 1 delta protein disulfide isomerase family A member 6 prolyl 4-hydroxylase subunit beta eukaryotic translation elongation factor 1 gamma eukaryotic translation elongation factor 1 beta 2 glycyl-tRNA synthetase proteasome 26S subunit, non-ATPase 11

52759 14144 74031 51810

1544 61 1672 677

74 3 84 28

18 2 24 7

3.58 0.8 3.68 1.08

1 1 1 1

10.693 10.426 9.938 9.035

2.994 3.389 3.34 4.109

10.199 15.035 14.285 13.715

24040 25805

509 73

33 4

9 2

3.78 0.38

1 1

8.062 8.017

1.127 2.123

10.449 9.389

31153 20981 32538 31410 39371 56366 30050 21707 63376 31011 40506 107629

185 1147 184 222 718 213 71 118 1341 216 304 81

5 68 9 7 30 6 3 4 68 6 13 4

2 12 4 3 9 2 2 1 21 4 4 2

0.31 12.3 0.68 0.49 2.24 0.16 0.32 0.47 3.96 0.72 0.52 0.08

1 1 1 1 1 1 1 1 1 1 1 1

7.761 7.057 5.958 5.871 5.417 5.104 4.94 4.934 4.814 4.58 4.522 4.482

3.184 1.087 1.134 2.658 3.917 3.875 2.688 2.402 1.759 2.148 2.277 3.176

10.482 9.127 8.174 8.098 7.492 6.828 8.163 5.586 8.086 7.169 4.05 6.222

29759 74428

243 212

6 4

3 3

0.53 0.19

1 1

4.442 4.273

3.412 3.088

6.855 6.434

52414

104

3

2

0.17

1

3.912

2.649

4.578

63998 54843

177 201

4 7

3 3

0.22 0.26

1 1

3.726 3.689

2.24 2.688

5.135 5.502

28063

258

10

3

0.57

1

3.305

1.857

4.1

90750 52622

115 55

3 2

2 2

0.1 0.17

1 1

3.12 3.036

2.445 2.269

4.069 5.642

a

(A) Possible new candidate Cad11-interacting proteins. (B) Candidate Cad11-interacting proteins identified by iTRAQ approach that are commonly present in mass spectrometry analyses. bemPAI: exponentially modified protein abundance index.

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 nonspecific 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 detects 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.

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 Brij-35.

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 356


Article


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.

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, that is, β-catenin, from the cytosol using the E-cadherin 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 predepleted with the cytoplasmic domain of E-cadherin.11 Given that βcatenin and angiomotin binding sites on Cad11 are adjacent to each other, this juxtaposition has led us to hypothesize that βcatenin 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 lowaffinity 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-tomesenchymal 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 Whereas it will be interesting to compare Cad11-interacting proteins in osteoblasts versus tumor cells, the Cad11-interacting proteins in mouse osteoblasts are unknown because we isolated the osteoblasts from mouse calvaria. 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 to mass spectrometry, the

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jproteome.7b00599. 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 analyses of proteins in Cad11 immune complexes from Brij-35, DDM, cholate, or Triton-X-100-solubilized PC3-mm2 cells.(XLSX) Supplemental Table captions. (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Fax: 713-834-6084. E-mail: [email protected]. ORCID

Sue-Hwa Lin: 0000-0002-6122-4054 Author Contributions

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. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS 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.

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REFERENCES

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Impact of Detergents on Membrane Protein Complex Isolation

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