Four-Dimensional Orthogonal Electrophoresis System for Screening
Protein Complexes and Protein-Protein Interactions Combined with
Xiaodong Wang, Guoqiang Chen, Hui Liu, Zhiyun Zhao, and Zhili Li*
Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and School of Basic Medicine,
Peking Union Medical College, Beijing 100005, China
Received June 10, 2010
Most current approaches for purification and identification of protein complexes adopt affinity
purifications combined with mass spectrometry, such as co-immunoprecipitation and tandem affinity
purification. Herein, we propose a new approach, termed as the four-dimensional orthogonal
electrophoresis (4-DE) system, to find and analyze the cytoplasmic protein complexes. 4-DE system is
composed of two parts: nondenaturing part (Part I) and denaturing part (Part II). Through Part I and
decision procedure separations, six protein complex candidates 20S core particle of proteasome (CP),
hemoglobin (Hb, R2?2), Hb (R2δ2), peroxiredoxin-2 (PRDX2), carbonic anhydrase-1 (CAH1), and heat
shock protein 60 (HSP60) were separated. CP, Hb (R2?2), PRDX2, and HSP60 with different MWs and
pI’s were chosen for Part II proteomic analysis. The results indicate that 4-DE is not only suitable for
studying protein complexes and protein-protein interactions as well as structural proteomics from
complex biological samples, but can also be easy to separate and concentrate intact protein complexes
from dilute complex samples.
Keywords: protein complex • proteomics • four-dimensional orthogonal electrophoresis • thin layer
IEF • mass spectrometry • protein-protein interaction
Protein complexes play very important roles in cells.1Almost
every major cellular process is carried out by protein com-
plexes. For instance, proteasome is involved in protein degra-
dation pathway,2Fanconi anemia complex participates in DNA
repair pathway,3and ribosome is necessary for protein syn-
thesis.4Although many protein complexes are well understood
especially in model organisms, such as Saccharomyces cerevi-
siae, most complexes in cells still remain unknown. Therefore,
to find, isolate, and characterize protein complexes more clearly
are the key factors to understand the essence of life.5,6
There are several traditional approaches which can be
applied to protein complex isolation and characterization, such
as ultracentrifugation, selective precipitation, multistep liquid
chromatography, and 2-DE. However, it has been reported that
these methods are not suitable for diluted samples,7and many
approaches suffer from time-consuming procedures and pos-
sibility of sample loss or subunit dissociations.8,9Alternatively,
as a brief procedure, affinity purifications may be the preferred
choice, such as co-immunoprecipitation (co-IP) and tandem
affinity purification (TAP). Co-IP has been used widely to isolate
and purify protein complexes.10TAP is well-known as a
powerful tool for purifying protein complexes under mild
conditions based on affinity for protein A and a calmodulin-
binding peptide (CBP) in the TAP tag.11Affinity purification
approaches are undoubtedly very effective for protein complex
study, but they have some disadvantages which limit their
applications. For example, TAP strategy is not a choice of
purification tool for endogenous protein complexes, and the
nonspecificity of antibody could bring in contaminant proteins.
Moreover, the high cost of antibody may hinder their use in
the cases of scaled-up purifications. Another shortcoming of
these approaches is that the purified proteins are usually
diluted and denatured. It is worth noting that high resolution
clear native electrophoresis (hrCNE) may be the preferred
choice for studying protein complexes described by Wittig et
al. recently.12This technique has been successfully applied for
the separation of physiologically active mitochondrial com-
plexes and Neisseria meningitidis outer membrane vesicle
Alternatively, isoelectric focusing (IEF) is not only suitable
for diluted protein samples, but can also be applied to separate
and concentrate protein complexes under mild conditions. The
IEF in the absence of denaturants such as urea and detergents
is called nondenaturing IEF or native IEF.15Recently, nonde-
naturing IEF has been used successfully in human plasma
proteins and protein complex studies, which were based on
capillary column gel or tube gel.16–19Herein, we described a
new approach, termed as the four-dimensional orthogonal
electrophoresis (4-DE) system, to find and analyze the protein
complexes from human cells (Figure 1), employing nondena-
* To whom correspondence should be addressed. Prof. Zhili Li, Institute
of Basic Medical Sciences, Chinese Academy of Medical Sciences and School
of Basic Medicine, Peking Union Medical College, 5 Dong Dan San Tiao,
Beijing 100005, China. E-mail: email@example.com. Phone: +86-10-
65296479. Fax: +86-10-65263815.
2010 American Chemical Society
Journal of Proteome Research 2010, 9, 5325–5334 5325
Published on Web 08/06/2010
turing thin layer IEF (tl-IEF) as the first dimension (1st-DE),
native polyacrylamide gel electrophoresis (native-PAGE) as the
second dimension (2nd-DE), denaturing IEF as the third
dimension (3rd-DE), and sodium dodecyl sulfate-polyacryl-
amide gel electrophoresis (SDS-PAGE) as the fourth dimension
(4th-DE). Alternatively, this 4-DE system is composed of two
parts: nondenaturing electrophoresis part (Part I) and denatur-
ing electrophoresis part (Part II). Part I includes the 1st-DE and
2nd-DE (1st-/2nd-DE), acting to find and purify the protein
complex candidates under mild conditions. The combination
of 1st-DE and 2nd-DE increases resolution, compared with the
only one-dimensional electrophoresis, nondenaturing tl-IEF,
or native-PAGE. Decision procedure includes MS analysis and
SDS-PAGE separation in order to identify protein complex
candidates and initially speculate their compositions. The
combination of the 3rd-DE and 4th-DE (3rd-/4th-DE) is Part
II, where the protein complex subunits would be separated.
Finally, we successfully found six protein complexes: 20S core
particle of proteasome (CP), hemoglobin (Hb, R2?2), Hb (R2δ2),
peroxiredoxin-2 (PRDX2), and carbonic anhydrase-1 (CAH1)
from erythrocytes and heat shock protein 60 (HSP60) from Raji
cells using Part I and decision procedure. Considering universal
applicability and stability of this system, the protein complex
candidates with different MWs and pI’s were chosen for further
analyses. CP (higher MW, medium pI), Hb (R2?2) (lower MW,
higher pI), PRDX2 (lower MW, lower pI), and HSP60 (medium
MW, lower pI) were further separated by Part II for proteomic
analysis, as the range of MWs and pI’s of the chosen protein
complexes had already covered CAH1. To inspect the stability
of this system, the medium MW complex HSP60, from linear
gradient gel, was chosen instead of Hb (R2δ2) from 5.5%
separating gel. Hb (R2δ2) and CAH1 were analyzed by SDS-
PAGE after extraction to inspect the efficiency of alkaline-ultra-
sonic and solution extraction. The results indicate that this
approach is easy to carry out and scale-up, making it suitable
for profiling of protein complexes and protein-protein interac-
tions, as well as structural proteomics.
Materials and Methods
Materials and Reagents. Erythrocytes were obtained from
donor blood via Ficoll-Paque density gradient centrifugation.20,21
Raji cell line was cultured in RPMI 1640 containing 10% (v/v)
fetal bovine serum. Ampholines (pH 3.0-9.5, 4.0-6.0, 5.0-7.0,
6.0-9.0) were obtained from the Academy of Military Medical
Sciences (Beijing, China). Succinyl-Leu-Leu-Val-Tyr-7-amino-
4-methylcoumarin (suc-LLVY-AMC) was obtained from BIO-
MOL (Plymouth Meeting, PA). R-Cyano-4-hydroxycinnamic
acid (CHCA) was from Sigma-Aldrich (St. Louis, MO). Phar-
malyte IPG buffer 3.0-10.0 was from GE Health Care (Freiburg,
Germany). Protease inhibitor cocktail tablets were obtained
from Roche Applied Science (Indianapolis, IN). Sequencing-
grade trypsin was purchased from Roche diagnostics (Man-
nheim, Germany). All other chemicals were obtained from
Merck (Darmstadt, Germany).
Erythrocyte Cytoplasmic Extracts. Erythrocytes were lysed
in nondenaturing lysis buffer (40 mM Tris-HCl, pH 7.5, 5 mM
MgCl2, 0.5 M NaCl, 1 mM dithiothreitol (DTT), 1 mM phenyl-
methanesulfonylfluoride (PMSF), 10 mM sodium fluoride, and
10 mM sodium pyrophosphate). The HMW cytoplasmic extracts
were obtained from erythrocyte lysis by three-step differential
centrifugations as reported previously.22Glycerol was added
at a final concentration of 40% (v/v) and the HMW cytoplasmic
extracts were then stored at -20 °C until use.
Raji Cytoplasmic Extracts. Raji cytoplasmic extracts were
prepared by incubating the Raji cells in hypotonic buffer (10
mM Tris, pH 7.4, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT,
and 2% (w/v) protease inhibitor cocktails) at 4 °C. After 30 min,
NP-40 (0.1% (v/v) final concentration) was added and the
nuclei were pelleted by centrifugation at 6000g for 10 s. The
cytoplasmic fraction was desalted and concentrated by Mi-
crosep Centrifugal Devices (3.0 kDa MWCO, Pall). It was then
transferred to a new tube and stored at -20 °C until use.
Protein Concentration Assay. The total concentration of
cytoplasmic extracts was determined by Braford assay,23using
bovine serum albumin (BSA) as a standard protein. The protein
concentrations of erythrocyte HMW cytoplasmic extracts and
Raji cytoplasmic extracts were 10.0 and 2.5 µg/µL, respectively.
The 1st-DE: Nondenaturing tl-IEF. Previous studies em-
ployed capillary column or tube gels as the first-dimensional
electrophoresis, providing relatively higher resolution and
shorter separation time for trace sample.15–19In this study, thin
layer gel was used instead of column or tube gel for the first-
dimensional electrophoresis, compatible with the IEF demands
of both resolution and larger protein amount loaded. Briefly,
4% (w/v) thin layer gel (130 mm × 100 mm × 1.0 mm,
acrylamide/bis-acrylamide ) 20:1) was employed for IEF in the
absence of denaturants such as urea and detergent, containing
Ampholines at pH 3.0-9.5, 4.0-6.0, 5.0-7.0, and 6.0-9.0 in
final concentrations of 1.0, 0.50, 0.50, and 0.50% (v/v), respec-
tively; 0.05% (v/v) TEMED and 0.05% (w/v) ammonium per-
sulfate (APS) were used as catalysts. As shown in Figure 2A,
the IEF was carried out at 150 V for 30 min as prefocusing step
(S1). Subsequently, the cytoplasmic extracts were loaded onto
the gel with IEF Sample Application Pieces (GE Healthcare,
Uppsala, Sweden). The IEF was carried out at 100 V for 30 min
as sample loading step (S2), followed by a stepwise voltage from
200 to 450 V (30 min and 50 V as interval time and voltage,
respectively) in midfocusing steps (S3-S8). In the final step
(S9), 500 V was kept until the current decreased to around 2.4
mA. All the steps described above were carried out at 4 °C. A
mixture of colored dyes was selected as the pI markers (BD
Bioscience, NJ). When the 1st-DE was finished, the focusing
gel was cut into strips along the edge of sample lanes. One of
the strips was stained using Coomassie G-250 (CBB), and
scanned by Umax powerlook 2100XL Scanner (Dallas, TX).
Other unstained strips were prepared for the 2nd-DE. After the
1st-DE separation, the IEF Sample Application Pieces were
recovered to determine the residual protein. The result indi-
cated that about 92% protein could be loaded into tl-IEF gels.
The 2nd-DE: Native-PAGE. After the 1st-DE, the unstained
strips were transferred into equilibrium buffer solution (0.01
M Tris/0.076 M glycine, pH 8.3), and were equilibrated for
Figure 1. The scheme of four-dimensional orthogonal electro-
phoresis (4-DE) system for screening of protein complexes and
protein-protein interactions combined with mass spectrometry.
Wang et al.
5326Journal of Proteome Research • Vol. 9, No. 10, 2010
30-45 min. Then, these gel strips were placed into the slot of
two glass plates (around 10 mm away from the top of glass
plates). For the erythrocyte cytoplasmic extracts, 5.5% (w/v)
separating gel (acrylamide/bis-acrylamide ) 20:1) was used as
described by Elsasser24with stacking gel. For the Raji cyto-
plasmic extracts, linear gradient gel (4-17%) was selected as
the separating gels as described by Margolis and Kenrik.25
When the separating gel solidified (45-60 min), 4% (w/v)
stacking gel was overlaid, in which the focusing gel strips were
also imbedded. All the native-PAGE gels were formed at room
temperature in a thin layer gel assembling cassette (130 mm
× 100 mm × 1.0 mm), and 25 mM Tris/192 mM glycine was
used as cathode and anode buffer. Electrophoresis was run at
10 mA/gel for 1 h, followed by 20 mA/gel for 5 h at 4 °C. HMW
native protein mixture (66-669 kDa) (GE Healthcare, Uppsala,
Sweden) was taken as molecular weight marker. The 2nd-DE
thin layer gels were stained by CBB, and then stored in 7%
(v/v) acetic acid at 4 °C until use. In this section, almost all the
protein (∼100%) in the 1st-DE gels could be transferred into
the 2nd-DE gels.
Alkaline-Ultrasonic and Solution Extraction of Protein
Complex Candidates from the 2nd-DE Gels. The spots of CBB
visualized protein complex candidates were cut along their spot
edges from the parallel 2nd-DE gels, and were then cut into
about 2-3 mm3pieces. Each protein complex candidate gel
piece was transferred into a new tube, followed by destaining
in 50% (v/v) acetonitrile (ACN)/25 mM NH4HCO3and washing
in water for 2 min, three times. The gel pieces were manually
crushed into about 0.2-0.3 mm3pieces. The tube was centri-
fuged briefly, and the supernatant was removed. A 200 µL
aliquot of 0.01 M NaOH solution was added and the tube was
sonicated for 5 min at 25 °C to extract the protein. The
supernatant was removed to a new tube, and the remaining
gel particles were washed with 200 µL of water thrice. The
supernatants were then pooled in the same tube. A 2 µL aliquot
of 1.0 M HCl was added to neutralize the solution. Finally, the
total supernatants were concentrated to the volume of around
50 µL using a SpeedVac vacuum concentrator and stored at
-20 °C until use. Five microliters of this concentrated liquid
could be used to carry out SDS-PAGE separation. To maximize
the recovery of protein from gel, the gel particles after the
alkaline-ultrasonic extraction were carried through a second
extraction by denaturing cocktail. The gel particles were
incubated in 450 µL of denaturing cocktail (7 M urea, 2 M
thiourea, and 2% (w/v) CHAPS) for 2 h or overnight at room
temperature, followed by extraction with 300 µL of water thrice.
The supernatants were pooled and concentrated to the volume
of around 420 µL using a SpeedVac vacuum concentrator. The
concentrated liquid mixed with the alkaline-ultrasonic ex-
tracted solution (45 µL) was to be subjected to the 3rd-/4th-
DE. More than 80% of the protein complexes could be
recovered from the 2nd-DE gels.
Figure 2. Nondenaturing tl-IEF (1st-DE) voltage optimization. (A) The procedure of voltage for the 1st-DE: step1, 150 V; step 2, 100 V;
step 3, 200 V; step 4, 250 V; step 5, 300 V; step 6, 350 V; step 7, 400 V; step 8, 450 V, and step 9, 500 V. (B-E) The curves of the current
versus run time were plotted. The 1st-DE was repeated for eight times and the current variations were recorded during the prefocusing
step (S1), sample loading step (S2), midfocusing steps (S3-S8), and final focusing step (S9), respectively.
4-DE System for Screening Protein Complexes
Journal of Proteome Research • Vol. 9, No. 10, 2010
SDS-PAGE Separation. SDS-PAGE separation was based on
the approach as described by Manabe17with slight modifica-
tion. Briefly, 5 µL aliquot of each protein complex candidate
extraction was treated with reduction-alkylation before being
loaded onto the SDS-PAGE gels based on the following steps.
First, 0.55 µL of 200 mM DTT in 250 mM NH4HCO3was added
into the extract solution (final DTT concentration of 20 mM),
and the solution was kept at 25 °C for 30 min. Then 0.62 µL of
500 mM iodoacetamide (IAA) in 25 mM NH4HCO3was added
(final IAA concentration of 50 mM) and the solution was
similarly kept at room temperature for 30 min. A 12% (w/v)
separating gel of SDS-PAGE was also performed at room
temperature using assembling cassette (130 mm × 100 mm ×
1.0 mm) (acrylamide/bis-acrylamide ) 30:1, 380 mM Tris-HCl,
pH 8.8, 1% (w/v) SDS), and then covered by 5% (w/v) stacking
gel (acrylamide/bis-acrylamide ) 30:1, 126 mM Tris-HCl, pH
6.8, 1% (w/v) SDS). Each preprepared protein solution mixed
with 1.6 µL of 5× SDS-PAGE loading buffer was loaded into
the well, and electrophoresis was run at 10 mA/gel for 30-45
min, followed by 20 mA/gel until the bands of bromophenol
blue migrated to the bottom of the gels. PageRuler Prestained
protein Ladder (SM0671) (Fermentas, Canada) was used in this
section as molecular weight marker.
The 3rd-/4th-DE: Denaturing IEF/SDS-PAGE. The solution
mixture of the alkaline-ultrasonic and solution extraction was
subjected to the 3rd-/4th-DE. Prior to IEF, IPG buffer (pH
3.0-10.0, nonlinear, GE Healthcare) and Destreak (GE Health-
care, Piscataway, NJ) were added at the final concentrations
of 1.2 and 0.5% (v/v), respectively. IPG DryStrips (pH 3.0-10.0,
nonlinear, 24 cm, GE Healthcare) were applied to Ettan
IPGphor 3 IEF System (GE Healthcare). After rehydration for
20 h at 40 V at 20 °C, IEF was conducted for 2 h each at 100,
200, 500, 1000 V, and 2 h at 1000-8000 V in gradient mode,
followed by 8000 V for a total of 144 kVh. Following IEF
separation, the gel strips were equilibrated for 15 min in the
buffer containing 50 mM Tris-HCl, pH 6.8, 7 M urea, 30%
(v/v) glycerol, 2% (w/v) SDS, 1% (w/v) DTT, and a trace of
bromophenol blue, followed by 15 min in the same buffer
except that 2.5% (w/v) IAA was used instead of 1% (w/v) DTT.
The fourth dimension of the separation (SDS-PAGE) was carried
out on Ettan DALTsix System (GE Healthcare) at 12% (w/v)
separating gel at 40 mA. Proteins were visualized by CBB. Gels
were scanned and stored at -20 °C until use. Overall, more
than 73% (∼92% × ∼100% × >80% based on the above
determination) of each protein complex could be applied in
this section, compared with those in the original samples.
In-Gel Fluorescent Assay of Hydrolysis Activity. To confirm
that the proteins or protein complexes separated by the 1st-/
2nd-DE remain biological active, the CP spot identified by
decision procedure (Supporting Information Table S1) was
selected for activity assay. The detailed procedure was similar
to that reported earlier.24Activity assay of the CP after extrac-
tion was also carried out with help of native-PAGE (Supporting
Information Figure S2). Briefly, CP spot was excised from the
2nd-DE and extracted by alkaline-ultrasonic and solution
extraction, followed by desalting and drying. Subsequently, it
was dissolved in refolding solution (50 mM Tris, 0.5 mM EDTA,
5 mM DTT, 5% glycerol, 25 mM MgCl2, 1 mM ATP, 0.2% PEG
4000, 1 mM GSH/2 mM GSSG, pH 8.0) overnight. The CP was
separated by 4-17% linear gradient native-PAGE before activity
In-Gel Protein Digestion. The CBB visualized spots of
protein complexes or proteins were excised from the 2nd-DE
or 4th-DE gels. For the 4th-DE, in-gel tryptic digestion was
carried out as previously reported.26For the 2nd-DE gel, the
gel pieces were destained and dehydrated, followed by 7 M urea
treatment for 1 h before the addition of 12.5 ng/µL trypsin in
25 mM NH4HCO3. Protein digestion was performed overnight
at 37 °C.
MS Analysis of Tryptic Peptides. MS analyses were per-
formed by the MALDI-TOF/TOF mass spectrometer (Autoflex
III, Bruker Daltonics, Billerica, MA). An aliquot of 1 µL of tryptic
digest, obtained from the 2nd-DE or 4th-DE gels, was spotted
onto MALDI target plate, and was dried at room temperature
before the addition of 1 µL of a saturated solution of CHCA in
70% (v/v) ACN/0.1% (v/v) TFA. MALDI-TOF spectra were
acquired in reflection mode. The peptide calibration standard
II mixture (Bruker Daltonics) was used for external calibration.
Trypsin autolysis peak (m/z 2163.05) was selected for internal
calibration. FlexAnalysis 3.0 software (Bruker Daltonics) was
used for subsequent data processing and peak lists generation.
Data mining was performed using BioTools 3.2 software (Bruker
Daltonics) with the following parameters: Taxonomy, Homo
sapiens; Database, Swiss-Prot; Enzyme, Trypsin; the global
carbamidomethylation of cysteine; the variable oxidation of
methionine and N-terminal acetylation were allowed for
database search; mass tolerance were set to 50 ppm for both
precursor ion and product ion spectra. All spectra were
Results and Discussion
Optimization of the 1st-DE Conditions. Previous studies
have shown that capillary column or tube gels could provide
relatively higher resolution within a shorter separation time as
nondenaturing IEF or denaturing IEF for the trace samples.27–29
In the present study, the thin layer gel was used instead of
capillary column or tube gel, compatible with the IEF demands
of both resolution and larger amounts of protein loaded.
Herein, the 1st-DE was regarded as a basal separation system,
as well as semipreparative or preparative tool for subsequent
separations. Therefore, the optimization of 1st-DE condition
is indispensable for the 4-DE system. The optimal conditions
are described as follows.
(i) The chemical composition of thin layer gel was prepared
similarly as described by Manabe.30The thin layer gel was used
as IEF gel including four Ampholines (pH 3.0-9.5, 4.0-6.0,
5.0-7.0 and 6.0-9.0) without denaturants such as urea or
detergents to enhance the gel resolution.
(ii) As shown in Figure 2A, the voltage and run time for 1st-
DE were optimized. The detailed process is described in
Materials and Methods. It is noteworthy that a pH gradient of
the tl-IEF gel should be formed at the prefocusing step, which
increases the pH difference between loaded proteins and gel,
so that sample is easily loaded into IEF gel (∼92% protein could
run into the gels based on the above determination). The
voltage change from 150 to 100 V in S2 step allowed the loaded
protein to more effectively run into gel under relatively low
voltage and be preserve in nondenaturing state. In midfocusing
steps, the stepwise voltages were adopted in order to shorten
the focusing time.
(iii) The stability and reproducibility of 1st-DE were tested.
The 1st-DE was carried out eight times based on the (ii) process
and the current values were recorded. The curves of current
versus time were plotted as shown in Figure 2B-E. It should
be noted that the current fluctuations were rather small,
Wang et al.
5328Journal of Proteome Research • Vol. 9, No. 10, 2010
suggesting that the stability and reproducibility of the thin layer
IEF gels were good.
(iv) The final focusing step of 1st-DE plays an important role
in the whole tl-IEF procedure. As shown in the Figure 3A, the
CP was in focusing process when the current value was >2.4
mA, and cathode drift took place when the current value was
<2.3 mA. The current between 2.3 and 2.4 mA as shown in
Figure 3B was assigned as the optimal current region where
the focusing procedure should be terminated, to avoid the
occurrence of overfocusing.
Protein Extraction from Polyacrylamide Gel. Herein, an
improved approach to shorten the extraction time and enhance
the recovery of protein from polyacrylamide gel was employed
by combining modified alkaline extraction with ultrasonic
methods based on previous studies.31,32As shown in Figure 4,
BSA was employed to optimize the concentration of NaOH and
extraction time. The experimental results indicated that the
recovery is related to the concentrations of NaOH (Figure 4A,B),
and the preferential concentration of NaOH for protein extrac-
tion was assigned as 0.01 M (the recovery was about 77.7%).
The increase of protein recovery was not significant when the
NaOH concentration was >0.01 M as shown in Figure 4B.
Evident in Figure 4C, it took only 5 min to extract most of
protein under ultrasonic condition (the recovery was about
80.4%). It is noteworthy that previous MS analyses indicated
that the alkaline cleavage of peptide bond was rather re-
stricted,31,32and the first-order rate constants of the ?-elimina-
tion reactions of protein covalent bonds caused by alkaline at
the most susceptible site were less than 0.1/h in 0.1 M NaOH
solution,31indicating that the protein degradation can be
negligible within the extraction time of 5 min in 0.01 M NaOH,
which was proved by Part II analysis.
To maximize the recovery of protein complexes from gel,
the gel particles after the alkaline-ultrasonic extraction were
subjected to solution extraction. After being mixed with
denaturing cocktail extraction solution, the HCl-neutralized
alkaline-ultrasonic solution ensures the concentration of NaCl
to be <5.0 mM, and this salt concentration was assumed to be
compatible with IEF without the desalting.
4-DE System. Recently, nondenaturing micro-2-DE was
reported by Manabe to analyze the human serum protein
complexes and protein-protein interactions.15,17–19In the
present study, we developed a 4-DE system to analyze the
protein complexes and protein-protein interactions as shown
in Figure 1. Briefly, 4-DE consisted of two parts: Part I and Part
II. Part I includes the 1st-DE and 2nd-DE (Figure 5A,C). Protein
Figure 3. The terminal time optimization of 1st-DE final step. (A) CP spots were stained by CBB on the 2nd-DE. (B) The mean current
versus time curve of eight pre-tests of 1st-DE was plotted. The CP was in focusing process when the current value was >2.4 mA, and
cathode drift took place when the current value was <2.3 mA. The current between 2.3 and 2.4 mA was assigned as the optimal range
in which the focusing procedure should be terminated, avoiding the occurrence of overfocusing (pink region).
Figure 4. The protein extraction recoveries were evaluated
through re-electrophoresis of BSA, which was extracted from
CBB-stained polyacrylamide gels by alkaline-ultrasonic extrac-
tion. (A and B) BSA was extracted by different concentration of
NaOH solutions within 10 min ultrasonic extraction, respectively.
(C) BSA was extracted by 0.01 M NaOH within different ultrasonic
extraction time, and the extraction recoveries were determined
by re-electrophoresis. All of the data were average of four
independent experiments (10 µg of BSA was loaded as refer-
4-DE System for Screening Protein Complexes
Journal of Proteome Research • Vol. 9, No. 10, 2010
complexes and interactional proteins were separated in Part I.
The fact that CP activity remains after sequential 1st-/2nd-DE
separations was monitored by the excitation at 365 nm using
suc-LLVY-AMC as CP substrate (Figure 5B,D), indicating that
Part I could keep protein complexes intact. However, activity
assay indicated that none of the CP activity was found (Sup-
porting Information Figure S2), suggesting that the denaturing
influences caused by alkaline-ultrasonic and solution extrac-
tion, CBB staining procedure, and reduction-alkylation for
protein complexes refolding might be irreversible. Decision
procedure includes MS analysis and SDS-PAGE separation.
Spots were excised from the 2nd-DE gels, denatured, and
subjected to in-gel tryptic digestion,26followed by the MS
identification (Figure 5E). Theoretical and experimental MWs
of the corresponding spots were calculated and compared with
each other to determine the protein complex candidates. The
subunits and interaction proteins of candidates were further
resolved by SDS-PAGE. If protein complex had shown more
than one band in gel, it might be heteromultimeric complex;
otherwise, it likely was homopolymer (Figures 5F, 6B, and 7B).
Part II is composed of the 3rd-DE and 4th-DE for profiling of
protein complexes, which were found in Part I and decision
procedure (Figures 5G, 6C, and 7C). Finally, we successfully
separated six protein complexes: CP, Hb (R2?2), Hb (R2δ2),
Figure 5. Establishment of 4-DE system with the help of CP. (A) HMW cytoplasmic extracts of erythrocytes were solved on the 4% (w/v)
1st-DE: nondenaturing tl-IEF, a mixture of colored dyes was selected as the pI markers. (B and D) The activity of CP on the 1st-DE and
2nd-DE gels were assayed using fluorogenic substrates suc-LLVY-AMC in the presence of 0.02% (w/v) SDS to confirm the proteolytic
activity (up), followed by subsequent CBB staining (down). (C) The 5.5% (w/v) 2nd-DE: native-PAGE was carried out after the 1st-DE.
CP was identified by MALDI-TOF MS (Supporting Information Table S1). (E) An example of MS/MS spectrum of tryptic sequence of R3,
LSAEKVEIATLTR, is listed. Expansion of the region of m/z 210-400 was displayed. (F) CP extracted by alkaline-ultrasonic extraction
from the 2nd-DE gels was subjected to 12% (w/v) SDS-PAGE detection, resulting in a characteristic pattern of CP bands of MW range
of 20-30 kDa. (G) A total of 10 spots of CP in parallel 2nd-DE gels were excised, and alkaline-ultrasonic protein extraction was performed
combined with solution extraction, followed by the 3rd-/4th-DE: denaturing IEF/SDS-PAGE. The CP subunits were visualized by silver
Wang et al.
5330Journal of Proteome Research • Vol. 9, No. 10, 2010
PRDX2, and CAH1 from the HMW cytoplasmic extracts of
human erythrocytes and HSP60 from the cytoplasmic extracts
of Raji cells.
4-DE Profiling of CP, Hb, and PRDX2 from Erythrocytes
and HSP 60 from Raji Cells. The erythrocyte HMW cytoplasmic
extracts and the Raji cell cytoplasmic extracts were separated
by Part I, and protein spots were identified by MS. As a result,
six protein complex candidates, CP (spot 1), Hb (R2?2) (spot
2, spot 3), Hb (R2δ2) (spot 4), PRDX2 (spot 5), CAH1 (spot 6),
and HSP60 (spot 1#) were determined (Figure 6A and 7A). On
the basis of the locations of those spots on the 2nd-DE gels,
experimental pI’s and MWs were estimated using the pI marker
and the HMW native protein mixture maker: CP (pI ≈ 5.8-6.3;
MW ≈ 668 kDa), Hb (R2?2) (spot 2) (pI ≈ 7.0; MW ≈ 180 kDa),
Hb (R2?2) (spot 3) (pI ≈ 7.1; MW ≈ 237 kDa), Hb (R2δ2) (pI ≈
7.5; MW ≈ 352 kDa), PRDX2 (pI ≈ 4.9; MW ≈ 159 kDa), CAH1
(pI ≈ 6.2; MW ≈ 442 kDa). and HSP60 (pI ≈ 4.4; MW ≈ 403
kDa) (Supporting Information Table S1). Considering universal
applicability and stability of this system, the protein complex
candidates with different MWs and pI’s were chosen for Part
II analysis. CP (higher MW, medium pI), Hb (R2?2) (spot 2)
(lower MW, higher pI), PRDX2 (lower MW, lower pI), and HSP60
(medium MW, lower pI) were further analyzed by Part II
proteomics analyses, as the range of MWs and pI’s of the
chosen protein complexes had already covered CAH1. To
inspect the stability of this system, the medium MW complex
HSP60, from linear gradient gel, was chosen instead of Hb
(R2δ2) from 5.5% separating gel. Hb (R2δ2) (spot 4) and CAH1
Figure 6. Purification and characterization of protein complexes from HMW cytoplasmic extracts of erythrocytes by 4-DE system combined
with MS. (A) HMW cytoplasmic extracts of erythrocytes were resolved by the 5.5% (w/v) 2nd-DE combined with the 1st-DE, and five
protein complex candidates were identified by MALDI-TOF MS (Supporting Information Table S1) and numbered on gels: CP (spot 1),
Hb (R2?2) (spot 2 and 3), Hb (R2δ2) (spot 4), PRDX2 (spot 5), and CAH1(spot 6). (B) CP, Hb (R2?2) (spot 2), and PRDX2 were chosen to
carry out SDS-PAGE detection after protein extraction. (C) In total, 20 spots of CP, 10 spots of Hb (R2?2) (spot 2), and 15 spots of
PRDX2 were excised from the parallel 2nd-DE gels, respectively, and alkaline-ultrasonic protein extraction combined with solution
extraction was performed, followed by Part II proteomics analysis. The protein complexes subunits were CBB visualized and characterized
by MS (Supporting Information Table S2).
4-DE System for Screening Protein Complexes
Journal of Proteome Research • Vol. 9, No. 10, 2010
(spot 6) were analyzed by SDS-PAGE after extraction to inspect
the efficiency of extraction (Supporting Information Figure S1).
The result indicated that Hb (R2δ2) and CAH1 could also be
separated by Part II for proteomics analyses, provided that the
number of corresponding spots was enlarged.
The CP is a hollow cylindrical structure composed of 28
subunits arranged in four stacked rings (R7?7?7R7). As shown
in Figure 6A, spot 1 was identified as CP subunits which is
consistent with previous report.33Because of the CP hetero-
geneity, which was reported previously by high-resolution free-
flow electrophoresis technology,34the pI of CP was focused in
a range from 5.8 to 6.3, indicating that the Part I may be utilized
as another simple and efficient approach for characterizing the
heterogeneity and/or subpopulation of protein complexes. The
SDS-PAGE and 3rd-/4th-DE (Part II) were performed for
subunit separation and mapping (Figure 6B,C). Forty-one spots
were identified unambiguously by MS, with at least one
identified peptide sequence (Supporting Information Table S2)
except for spot 4 (?1). The lack of ?5 might be due to the poor
focusing in the corresponding pI region on this gel. All of the
CP subunits were featured with at least two isoforms on the
3rd-/4th-DE map, excluding R5 and ?6. R5 was reported as only
one detectable isoform in human cells, such as erythrocytes,8,22
U937,9Caco-2, and HT-29.35The detection of ?6 with only one
isoform might be due to the poor focusing and/or separation.
The MW variation of ?7 and R1 isoforms was observed in Figure
6C (spots 9, 10 and spots 12, 13), which was likely due to the
mRNA splicing or proteolytic cleavages.8The fact that the
observed isoforms of a given subunit display shifted pI values
suggested that their presence was more likely the result of post-
translational modification (PTM) rather than proteolytic cleav-
ages. The human CP exhibited a high degree of structural
heterogeneity with the help of 4-DE system, which might
account for variations in catalytic activities observed for
As shown in Figure 6A, spots 2-4 were identified as Hb
complex, R2?2 assembly (spots 2, 3) and R2δ2 assembly (spot
4). Hb (R2?2), which is composed of two R subunits and two ?
subunits, occupies the dominant component in Hb of adult
humans. The amount of Hb (R2δ2) is less than that of Hb
(R2?2). The experimental MWs of spots 2, 3, and 4 were
calculated as 180, 237, and 352 kDa, respectively, comparing
with the theoretical MW (approximately 64.5 kDa for tet-
ramer)37of Hb. There was significant difference in MWs
between experimental and theoretical calculation. Probably, R
and ? or R and δ subunits could aggregate to form oligomer
larger than tetramer, indicating that the part I might be a soft
separate approach for protein complexes study. Spot 2 was
subjected to SDS-PAGE detection (Figure 6B) and the 3rd-/
4th-DE separation (Figure 6C). The existence of multi-subunits
Figure 7. Purification and characterization of HSP60 from Raji cytoplasmic extracts by 4-DE system combined with MS. (A) Raji
cytoplasmic extracts were resolved by the 4-17% (w/v) 2nd-DE after the 1st-DE, and HSP60 was identified by MALDI-TOF MS (Supporting
Information Table S1) and marked on gels. (B) HSP60 was chosen to carry out SDS-PAGE detection after protein extraction. (C) Fifteen
spots of HSP60 were excised from the parallel 2nd-DE gels, and alkaline-ultrasonic protein extraction combined with solution extraction
was performed before the 3rd-/4th-DE analysis. The subunit isoforms of HSP60 were visualized by CBB and identified by combined
PMF and MS/MS (Supporting Information Table S2).
Wang et al.
5332Journal of Proteome Research • Vol. 9, No. 10, 2010
can be proven in Figure 6B. As further profiled in Figure 6C,
13 Hb (?) and 4 Hb (R) isoforms mainly raised from PTMs,
proved by MS/MS, might act as major contributor to the
existence of many variants of Hb according to a previous
study.38The difference in MWs of Hb (?) isoforms was likely
due to the results of proteolytic cleavages and alternative
PRDX2 and HSP60 have been proven as ring-like homo-
polymers.39,40As shown in Figures 6A (spot 5) and 7A (spot
1#), the experimental MWs of PRDX2 and HSP60 were calcu-
lated as 159 and 403 kDa, respectively. SDS-PAGE detection
proved that PRDX2 and HSP60 were composed of monomers
(Figures 6B and 7B). Comparing with the theoretical MWs of
PRDX2 monomer (about 21.8 kDa) and HSP60 monomer (about
58.0 kDa), we could deduce that spot 5 and spot 1#on 2nd-DE
gels might represent the octamer and heptamer forms for
PRDX2 and HSP6, respectively, which is consistent with a
previous study.41,42The octamer of PRDX2 was confirmed by
Li et al.41It indicates that octamer of PRDX2 was also present
in human erythrocytes. The previous study proved that Hsp60
was an oligomer, composed of two stacked rings each built by
seven identical subunits, the Hsp60 monomers, under physi-
ological conditions.42The lack of tetradecamer of HSP60 in
Figure 7A might be due to unstability, comparing with hep-
tamer. The 3rd-/4th-DE was assigned to further separate the
heterogeneities of PRDX2 and HSP60 subunits. Four isoforms
of PRDX2 and eight isoforms of HSP60 were observed and
identified unambiguously by MS (Supporting Information Table
S2). As shown on the 3rd-/4th-DE gels (Figures 6C and 7C), all
of the PRDX2 isoforms exhibited clearly different pI values
without significant MW variations, suggesting that the differ-
ence was likely due to PTMs, rather than the consequence of
proteolytic cleavages and/or mRNA splicing events.43The
HSP60 isoforms displayed both pI and MW changes, which
might be originated from PTMs, proteolytic cleavages, mRNA
splicing events, and/or the derivatization of cysteine residues
with gel components.44
In this study, we successfully developed a 4-DE system for
proteomic analyses of human cytoplasmic protein complexes
and protein-protein interactions. First, we used tl-IEF instead
of capillary or tube IEF to enlarge the loading amounts of
protein samples, and the optimal condition of 1st-DE was
carried out for subsequent electrophoresis separations. We also
proposed an improved protein alkaline-ultrasonic extraction
approach combined with solution extraction from gels, in
which the extraction solution could be directly subjected to
the 3rd-DE separation without the need of desalting. More than
73% of each protein complex can be applied in Part II
proteomics analyses, compared with those in the original
samples. Through Part I and decision procedure separations,
six protein complex candidates: CP, Hb (R2?2), Hb (R2δ2),
PRDX2, CAH1, and HSP60 were found, four of which were
mapped by Part II. The results support and complement the
previous observations for unveiling the diversities of human
CP, Hb, PRDX2, and HSP60 subunit isoforms on the 4-DE
maps. Taken together, this 4-DE system is easy to carry out
for the separation, proteomic analyses, and subunit stoichi-
ometry studies of protein complexes even from complex
biological sample such as cell lysate.
Acknowledgment. This investigation was supported
by grant 2006AA02Z154 awarded by National High-Tech
R&D Program of China (863 Program) and grant 20675088
from the National Natural Science Foundation of China, and
also supported by grant 20070023023 awarded by the Ph.D.
Programs Foundation of Ministry of Education of China.
Supporting Information Available: Tables of the PMF
and MS/MS results obtained from the 2nd-DE and 4th-DE gels
(Table S1, list of protein complex candidates in the 2nd-DE
identified by PMF and MS/MS; Table S2, list of PMF and MS/
MS results obtained from the 4th-DE). This material is available
free of charge via the Internet at http://pubs.acs.org.
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