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*,†
†
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
*
See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

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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 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

■ 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 vitro6 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, 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
Received: August 23, 2017
Published: November 7, 2017

© 2017 American Chemical Society 348 DOI: 10.1021/acs.jproteome.7b00599

J. Proteome Res. 2018, 17, 348−358
Journal of Proteome Research
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.
■
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). Zwittergent 3-12, Brij-35, sodium cholate,
digitonin, and phenylmethylsulfonyl fluoride were from Sigma-
Aldrich (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).
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 electro-
phoresis 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
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transferred onto nitrocellulose membranes. For immunoblot-
ting, 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 chemilumines-
cence (ECL) (Thermo Fisher Scientific).
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 chromatog-
raphy−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
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%
DOI: 10.1021/acs.jproteome.7b00599
J. Proteome Res. 2018, 17, 348−358
Journal of Proteome Research
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. 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 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
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weighted, using only peptides with expectation values <0.05.
Because 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 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.
DOI: 10.1021/acs.jproteome.7b00599
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EDTA is also commonly included in the immunoprecipita-
tion 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 X-100, 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 solubi-
lized 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 immunopreci-
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 four times.
pitation 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 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
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conformational change that interferes with mAb 1A5 binding to
Cad11.
We further examined the effects of seven additional
detergents on Cad11 solubilization and immunoprecipitation
at a detergent to protein ratio of 5. The detergents used were
Brij-35, sodium cholate, Triton X-100, CHAPSO, Big CHAP,
Zwittergent 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 Brij-35-
solubilized fraction compared with other detergents (Figure
3A), suggesting that Brij-35 may not be able to solubilize PC3-
mm2 proteins efficiently or the detergent induces protein
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, and p120-catenin were solubilized by all
detergents tested except Brij-35. Clathrin was only detected in the total
cell lysates but not in detergent-solubilized lysates. GAPDH showed
that all detergents, except Brij-35, 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 Brij-35. However,
p120-catenin was only detected in immune complexes of Big CHAP
and digitonin but not in DDM, cholate, CHAPSO or Zwittergent 3-12.
These results suggest that these detergents may interfere with Cad11
and p120-catenin interaction.
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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 Brij-35, 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 detergent-
solubilized 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 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.
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 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
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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 two times.
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 T-
complex 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 DDM-
solubilized 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).
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
DOI: 10.1021/acs.jproteome.7b00599
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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 prot_desc prot_mass prot_score prot_matches prot_sequences prot_cover emPAI
CLH1_HUMAN CLTC clathrin heavy chain 1 191493 410, 240 19, 11 16, 9 16.1, 13.2 0.49, 0.25
CAD11_HUMAN CDH11 cadherin-11 87911 1615, 1385 80, 77 35, 35 42.7, 45.1 5.72, 5.74
CTNB1_HUMAN CTNNB1 catenin beta-1 85442 1792, 1683 73, 69 33, 33 54.8, 56 5.71, 6.12
PLAK_HUMAN JUP junction plakoglobin 81693 834, 880 39, 37 23, 24 36.9, 42.8 2.85, 3.09
TCPA_HUMAN TCP1 T-complex protein 1 subunit alpha 60306 40, 113 1, 4 1, 4 1.8, 14.2 0.08, 0.37
TCPB_HUMAN CCT2 T-complex protein 1 subunit beta 57452 47, 77 1, 2 1, 2 2.2, 10.8 0.09, 0.18
TCPD_HUMAN CCT4 T-complex protein 1 subunit delta 57888 59, 93 2, 4 2, 4 5.9, 11.5 0.18, 0.39
TCPE_HUMAN CCT5 T-complex protein 1 subunit 59633 44, 55 1, 1 1, 1 1.8, 9.2 0.08, 0.08
epsilon
TCPG_HUMAN CCT3 T-complex protein 1 subunit 60495 73, 126 1, 3 1, 3 2, 16 0.08, 0.27
gamma
TCPH_HUMAN CCT7 T-complex protein 1 subunit eta 59329 58, 51 2, 3 2, 3 4.4, 17.3 0.17, 0.27
TCPQ_HUMAN CCT8 T-complex protein 1 subunit theta 59583 43, 162 2, 5 2, 5 10, 23.7 0.17, 0.49
TCPZ_HUMAN CCT6A T-complex protein 1 subunit zeta 57988 71, 76 2, 2 2, 2 5.3, 9.6 0.18, 0.18
(B) Mass Spectrometry Analyses of Proteins in SDS-PAGE with Apparent Molecular Masses between 50 and 25 kDa from Duplicate Samples
prot_acc GN prot_desc prot_mass prot_score matches sequences prot_cover emPAI
CAD11_HUMAN CDH11 cadherin-11 87911 346, 354 19, 20 11, 14 19.7, 21.5 2.24, 0.72
CTNB1_HUMAN CTNNB1 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 voltage-dependent anion-selective channel 31547 117, 216 7, 8 6, 6 27.2, 27.2 0.23, 0.9
protein 2
CLCA_HUMAN CLTA clathrin light chain A 27060 44, 42 2, 2 2, 1 7.7, 3.6 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 SDS-
PAGE 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 symbol Entrez gene name mass score no. of matches no. of sequences emPAI Brij-35 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
C9J826 JUP junction plakoglobin 32204 72 5 2 0.3 1 7.57 4.46 7.00
P09496-2 CLTA clathrin light chain A 25377 106 2 2 0.39 1 7.38 2.98 6.45
A0A087WVQ6 CLTC clathrin heavy chain 206058 160 8 6 0.13 1 3.01 1.81 3.55
C9JZR2 CTNND1 catenin delta 1 112710 54 4 2 0.08 1 2.06 2.36 6.16
(B) Common Candidate Cad11-Interacting Proteins Identified by LC−MS/MS of SDS-PAGE Gel Slices and by iTRAQ Approach
no. of no. of Brij-
ID accession symbol Entrez gene name mass score matches sequences emPAI 35 DDM cholate TX100
J3KPX7 PHB2 prohibitin 2 36101 205 12 8 1.54 1 3.285 1.316 4.576
P17987 TCP1 t-complex 1 66214 291 13 6 0.47 1 5.395 3.563 8.891
P50991 CCT4 chaperonin containing TCP1 63652 294 11 7 0.59 1 4.357 2.78 6.419
subunit 4
B7ZAR1 CCT5 chaperonin containing TCP1 61222 110 4 4 0.32 1 6.614 4.309 8.987
subunit 5
Q99832 CCT7 chaperonin containing TCP1 65381 149 3 2 0.14 1 4.5 2.839 7.454
subunit 7
P50990 CCT8 chaperonin containing TCP1 65635 417 13 7 0.57 1 3.74 2.482 6.121
subunit 8
A0A0A0MR02 VDAC2 voltage dependent anion channel 2 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 complexes. PC3-mm2 cells were solubilized by DDM, sodium
of proteins in differentially solubilized Cad11 immune cholate, or Triton X-100 and were then immunoprecipitated by
353 DOI: 10.1021/acs.jproteome.7b00599
J. Proteome Res. 2018, 17, 348−358
Journal of Proteome Research
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 Brij-
35, 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
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-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
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
Article
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.
■ 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. Addi-
tionally, 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
DOI: 10.1021/acs.jproteome.7b00599
J. Proteome Res. 2018, 17, 348−358
Journal of Proteome Research Article

Table 3. Candidate Cad11-Interacting Proteins Identified by iTRAQ Approacha,b

(A) Possible New Candidate Cad11-Interacting Proteins Identified by iTRAQ Approach
no. of no. of Brij-
ID accession symbol Entrez gene name mass score matches sequences emPAI 35 DDM cholate TX100
P30086 PEBP1 phosphatidylethanolamine binding 23349 191 12 3 0.71 1 16.648 3.183 20.354
protein 1
P52758 RIDA reactive intermediate imine 15782 171 5 2 0.69 1 11.872 4.737 14.785
deaminase A homologue
F8WDS9 LANCL1 LanC like 1 10285 63 2 1 0.49 1 8.765 3.777 14.968
Q16658 FSCN1 fascin actin-bundling protein 1 58675 241 8 3 0.24 1 7.941 1.775 6.797
O60493 SNX3 sorting nexin 3 20336 59 2 2 0.51 1 7.696 2.628 13.458
A0A087X0R6 SNX12 sorting nexin 12 21254 68 3 2 0.48 1 7.432 2.346 11.748
P62942 FKBP1A FK506 binding protein 1A 13240 308 11 3 1.55 1 7.041 2.515 6.727
A0A087X0W7 ACOT2 acyl-CoA thioesterase 2 49001 93 3 2 0.19 1 6.245 2.799 5.621
P27797 CALR calreticulin 54308 89 3 2 0.17 1 5.799 2.659 7.099
K7ENL7 VASP vasodilator-stimulated 11746 63 2 1 0.42 1 5.723 1.752 5.346
phosphoprotein
Q15942 ZYX zyxin 65129 281 13 4 0.3 1 5.637 1.648 4.703
A0A087WWJ2 NAA50 N(alpha)-acetyltransferase 50, 11304 56 2 1 0.44 1 5.446 2.126 8.322
NatE catalytic subunit
P60709 ACTB actin beta 44592 2307 109 19 11.84 1 5.069 4.561 8.822
Q5JRA6 MIA3 MIA family member 3, ER export 235042 76 3 1 0.02 1 4.942 2.844 5.401
factor
P04899 GNAI2 G protein subunit alpha i2 44460 196 4 2 0.21 1 4.94 3.511 6.682
Q99714 HSD17B10 hydroxysteroid 28635 107 6 5 1.08 1 4.921 3.011 8.224
17-beta dehydrogenase 10
P55072 VCP valosin containing protein 96183 237 9 7 0.36 1 4.734 3.142 7.658
A0A0U1RQF0 FASN fatty acid synthase 285419 213 12 7 0.11 1 4.522 3.071 7.039
P07737 PFN1 profilin 1 16630 398 14 6 4.74 1 4.445 2.093 5.281
A0A087WTT1 PABPC1 poly(A) binding protein 64407 162 3 2 0.14 1 4.376 3.514 4.995
cytoplasmic 1
Q9BQ69 MACROD1 MACRO domain containing 1 38941 195 5 2 0.24 1 4.292 1.781 5.915
P22307 SCP2 sterol carrier protein 2 65728 315 17 7 0.79 1 4.059 1.092 6.484
P35579 MYH9 myosin heavy chain 9 256365 93 4 4 0.07 1 3.998 2.604 4.72
C9JFR7 CYCS cytochrome c, somatic 14064 204 9 5 3.34 1 3.989 1.403 5.594
P22314 UBA1 ubiquitin like modifier activating 125556 227 7 6 0.22 1 3.772 2.981 4.107
enzyme 1
P09669 COX6C cytochrome c oxidase subunit 6C 9929 50 2 1 0.51 1 3.716 2.596 4.066
H0YNE9 RAB8B RAB8B, member RAS oncogene 24448 155 8 3 0.67 1 3.713 3.088 6.817
family
H0YIZ0 SHMT2 serine hydroxymethyltransferase 2 30818 112 4 2 0.31 1 3.678 3.2 5.331
P16144 ITGB4 integrin subunit beta 4 211405 83 2 1 0.02 1 3.599 1.776 4.813
G5EA31 SEC24C SEC24 homologue C, COPII coat 116237 150 8 5 0.2 1 3.551 2.88 5.399
complex component
A0A0G2JLD8 SSBP1 single stranded DNA binding 16597 148 7 3 1.11 1 3.538 2.04 3.16
protein 1
P37802 TAGLN2 transgelin 2 24250 220 10 6 2.36 1 3.515 1.982 5.376
O95831 AIFM1 apoptosis inducing factor 72911 85 3 2 0.12 1 3.375 2.579 4.004
mitochondria associated 1
P06576 ATP5B ATP synthase, H+ transporting, 59983 748 32 10 1.68 1 3.277 2.928 5.488
mitochondrial F1 complex, beta
polypeptide
Q96HC4 PDLIM5 PDZ and LIM domain 5 69092 61 2 2 0.13 1 3.259 2.607 4.604
A2A274 ACO2 aconitase 2 95835 354 14 6 0.3 1 3.195 1.179 4.425
O15143 ARPC1B actin related protein 2/3 complex 44814 72 2 1 0.1 1 3.136 2.288 4.649
subunit 1B
K7ERT7 VAT1 vesicle amine transport 1 15770 104 2 1 0.3 1 3.116 2.919 4.673
P08574 CYC1 cytochrome c1 37560 118 2 1 0.12 1 3.036 2.5 4.505
P05556 ITGB1 integrin subunit beta 1 96715 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
no. of no. of Brij-
ID accession symbol Entrez gene name mass score matches sequences emPAI 35 DDM cholate TX100
P62937 PPIA peptidylprolyl isomerase A 20162 760 35 10 8.7 1 14.694 3.844 15.699
A0A0C4DFU2 SOD2 superoxide dismutase 2 27040 84 2 1 0.17 1 13.908 4.767 16.823
P30405 PPIF peptidylprolyl isomerase F 24476 566 30 7 2.3 1 11.744 4.493 14.961
P00338 LDHA lactate dehydrogenase A 40844 532 32 7 1.06 1 11.59 2.851 15.167

355 DOI: 10.1021/acs.jproteome.7b00599

J. Proteome Res. 2018, 17, 348−358
Journal of Proteome Research Article

Table 3. continued
(B) Candidate Cad11-Interacting Proteins That Are Commonly Present in Mass Spectrometry Analyses
no. of no. of Brij-
ID accession symbol Entrez gene name mass score matches sequences emPAI 35 DDM cholate TX100
P06733 ENO1 enolase 1 52759 1544 74 18 3.58 1 10.693 2.994 10.199
H0YKB3 SORD sorbitol dehydrogenase 14144 61 3 2 0.8 1 10.426 3.389 15.035
P29401 TKT transketolase 74031 1672 84 24 3.68 1 9.938 3.34 14.285
P00505 GOT2 glutamic-oxaloacetic 51810 677 28 7 1.08 1 9.035 4.109 13.715
transaminase 2
P32119 PRDX2 peroxiredoxin 2 24040 509 33 9 3.78 1 8.062 1.127 10.449
B4DLR8 NQO1 NAD(P)H quinone 25805 73 4 2 0.38 1 8.017 2.123 9.389
dehydrogenase 1
P25788 PSMA3 proteasome subunit alpha 3 31153 185 5 2 0.31 1 7.761 3.184 10.482
A0A0A0MSI0 PRDX1 peroxiredoxin 1 20981 1147 68 12 12.3 1 7.057 1.087 9.127
Q13162 PRDX4 peroxiredoxin 4 32538 184 9 4 0.68 1 5.958 1.134 8.174
P25786 PSMA1 proteasome subunit alpha 1 31410 222 7 3 0.49 1 5.871 2.658 8.098
P40926 MDH2 malate dehydrogenase 2 39371 718 30 9 2.24 1 5.417 3.917 7.492
Q5SZU1 PHGDH phosphoglycerate dehydrogenase 56366 213 6 2 0.16 1 5.104 3.875 6.828
P28070 PSMB4 proteasome subunit beta 4 30050 71 3 2 0.32 1 4.94 2.688 8.163
I3L3B0 C1QBP complement C1q binding protein 21707 118 4 1 0.47 1 4.934 2.402 5.586
P14618 PKM pyruvate kinase, muscle 63376 1341 68 21 3.96 1 4.814 1.759 8.086
G3V5Z7 PSMA6 proteasome subunit alpha 6 31011 216 6 4 0.72 1 4.58 2.148 7.169
P07195 LDHB lactate dehydrogenase B 40506 304 13 4 0.52 1 4.522 2.277 4.05
Q13200 PSMD2 proteasome 26S subunit, 107629 81 4 2 0.08 1 4.482 3.176 6.222
non-ATPase 2
P28074 PSMB5 proteasome subunit beta 5 29759 243 6 3 0.53 1 4.442 3.412 6.855
A0A087X1X7 EEF1D eukaryotic translation elongation 74428 212 4 3 0.19 1 4.273 3.088 6.434
factor 1 delta
Q15084 PDIA6 protein disulfide isomerase 52414 104 3 2 0.17 1 3.912 2.649 4.578
family A member 6
P07237 P4HB prolyl 4-hydroxylase subunit beta 63998 177 4 3 0.22 1 3.726 2.24 5.135
P26641 EEF1G eukaryotic translation elongation 54843 201 7 3 0.26 1 3.689 2.688 5.502
factor 1 gamma
P24534 EEF1B2 eukaryotic translation elongation 28063 258 10 3 0.57 1 3.305 1.857 4.1
factor 1 beta 2
P41250 GARS glycyl-tRNA synthetase 90750 115 3 2 0.1 1 3.12 2.445 4.069
O00231 PSMD11 proteasome 26S subunit, 52622 55 2 2 0.17 1 3.036 2.269 5.642
non-ATPase 11
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

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
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 immunopreci-
pitation 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.
DOI: 10.1021/acs.jproteome.7b00599
J. Proteome Res. 2018, 17, 348−358
Journal of Proteome Research
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 demon-
strated 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 β-
catenin and angiomotin binding sites on Cad11 are adjacent to
each other, this juxtaposition has 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 Whereas it will be
interesting to compare Cad11-interacting proteins in osteo-
blasts 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 inter-
actions, leading to alterations in the composition of protein
complexes purified by immunoprecipitation. We have also
demonstrated that coupled to mass spectrometry, the
357
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.
■
*
ASSOCIATED CONTENT
S Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jproteo-
me.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)
■
AUTHOR INFORMATION
Corresponding Author
*Fax: 713-834-6084. E-mail: slin@mdanderson.org.
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.
■
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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358 DOI: 10.1021/acs.jproteome.7b00599

J. Proteome Res. 2018, 17, 348−358