RESEARCH ARTICLE

Analysis of the synaptic vesicle proteome using three

gel-based protein separation techniques
Jacqueline Burr
1
*
, Tobias Beckhaus
2
*
, Hermann Schgger
3
, Carsten Corvey
2
,
Sandra Hofmann
2
, Michael Karas
2
, Herbert Zimmermann
1
and Walter Volknandt
1
1
Department of Neurochemistry, Johann Wolfgang Goethe-University, Frankfurt/Main, Germany
2
Department of Pharmaceutical Chemistry, Johann Wolfgang Goethe-University, Frankfurt/Main, Germany
3
Molecular Bioenergetics, Johann Wolfgang Goethe-University, Frankfurt/Main, Germany
Synaptic vesicles are key organelles in neurotransmission. Their functions are governed by a
unique set of integral and peripherally associated proteins. To obtain a complete protein inven-
tory, we immunoisolated synaptic vesicles from rat brain to high purity and performed a gel-
based analysis of the synaptic vesicle proteome. Since the high hydrophobicity of integral mem-
brane proteins hampers their resolution by gel electrophoretic techniques, we applied in parallel
three different gel electrophoretic methods for protein separation prior to MS. Synaptic vesicle
proteins were subjected to either 1-D SDS-PAGE along with nano-LC ESI-MS/MS or to the 2-D
gel electrophoretic techniques benzyldimethyl-n-hexadecylammonium chloride (BAC)/SDS-
PAGE, and double SDS (dSDS)-PAGE in combination with MALDI-TOF-MS. We demonstrate
that the combination of all three methods provides a comprehensive survey of the proteinaceous
inventory of the synaptic vesicle membrane compartment. The identified synaptic vesicle pro-
teins include transporters, soluble N-ethylmaleimide-sensitive factor attachment protein recep-
tors (SNAREs), synapsins, rab and rab-interacting proteins, additional guanine nucleotide tri-
phosphate (GTP) binding proteins, cytoskeletal proteins, and proteins modulating synaptic
vesicle exo- and endocytosis. In addition, we identified novel proteins of unknown function. Our
results demonstrate that the parallel application of three different gel-based approaches in com-
bination with mass spectrometry permits a comprehensive analysis of the synaptic vesicle pro-
teome that is considerably more complex than previously anticipated.
Received: May 12, 2006
Revised: July 26, 2006
Accepted: August 12, 2006
Keywords:
Mass spectrometry / Membrane protein / Proteome / Synaptic vesicle protein / Two-
dimensional electrophoresis
6250 Proteomics 2006, 6, 62506262
1 Introduction
Synaptic vesicles represent organelles of the presynaptic
nerve ending that control essential functions of chemical
synaptic transmission [1, 2]. Vesicle integral membrane pro-
teins and vesicle-associated proteins are central players in
synaptic vesicle function. These include organelle transport,
its interaction with the cytoskeleton, uptake and storage of
neurotransmitters, and regulated exo- and endocytosis.
Within the past few years, converging work from several
laboratories resulted in a molecular and functional charac-
terization of major synaptic vesicle proteins [3]. Proteins
responsible for the mechanisms of neurotransmitter release
have been investigated in detail [4]. Proteins involved in the
Correspondence: Jacqueline Burr, Department of Neurochemis-
try, Johann Wolfgang Goethe-University, Max-von-Laue-Strasse
9, 60438 Frankfurt/Main, Germany
E-mail: j.burre@cns.uni-frankfurt.de
Fax: 149-69-798-29606
Abbreviations: -SNAP, ab-soluble NSF attachment protein;
AMPA, a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid;
BAC, benzyldimethyl-n-hexadecylammonium chloride; dSDS,
double SDS; GAPDH, glyceraldehyde 3-phosphate dehydrogen-
ase; GFAP, glial fibrillary acidic protein; gravy, grand average of
hydrophobicity index; NSF, N-ethyl maleimide sensitive factor;
PSD, postsynaptic density; SCAMP, secretory carrier-associated
membrane protein; SNAP-25, synaptosome-associated protein of
25 kDa; SNARE, soluble N-ethylmaleimide-sensitive factor attach-
ment protein receptor; SV2, synaptic vesicle protein 2; VAMP,
vesicle-associated membrane protein; vGaT, vesicular GABA
transporter; vGluT1, vesicular glutamate transporter; ZnT, zinc
transporter * Both authors contributed equally to this work.
DOI 10.1002/pmic.200600357
2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com
Proteomics 2006, 6, 62506262 Animal Proteomics 6251
docking and fusion process during exocytosis include the
soluble N-ethylmaleimide-sensitive factor attachment pro-
tein receptor (SNARE)-complex [5] as well as soluble proteins
involved in the functional regulation of this complex. Addi-
tional proteins are expected to modulate neurotransmitter
release and are of central importance for the numerous
functions the organelle fulfils during its life cycle.
Organellar proteomics has been limited by the biochem-
ical and biophysical techniques available. The development
of new protocols for the separation and identification of or-
ganelle proteins along with extensive protein data libraries
resulted in the characterization of the proteomes of a num-
ber of organelles, such as lysosomes, golgi stacks, and mito-
chondria [68]. Recently, two papers reported a first analysis
of the synaptic vesicle proteome [9, 10]. A recent report by
Blondeau et al. [11] identified a total of 209 proteins of clath-
rin coated vesicles, many of them assigned to the synaptic
vesicle compartment. In order to obtain a more complete
protein inventory of this organelle, we performed a gel-based
analysis of the entire synaptic vesicle proteome. Synaptic
vesicles are rich in proteins spanning the membrane several
times [4]. The hydrophobicity of these proteins hampers
their resolution by gel electrophoretic techniques. We,
therefore, applied in parallel three different gel electropho-
retic methods for protein separation prior to protein identi-
fication by MS. Synaptic vesicle proteins were subjected to
either 1-D SDS-PAGE [12] along with nano-LC ESI-MS/MS
or to the 2-D techniques BAC/SDS-PAGE [13], and double
SDS (dSDS)-PAGE [14], performed in combination with
MALDI-TOF-MS. The limitations of IEFfor the separation of
hydrophobic synaptic vesicle membrane proteins have been
described [9, and own observations], whereas the two 2-D
techniques used in this study have been previously opti-
mized for use with membrane proteins [13, 14]. BAC/SDS-
PAGE involves discontinuous gel electrophoresis in an acidic
buffer system using the cationic detergent benzyldimethyl-n-
hexadecylammonium chloride (16-BAC) in the first dimen-
sion and discontinuous SDS-PAGE in the second dimen-
sion. dSDS-PAGE is based on a low acrylamide gel for the
first-dimensional SDS-PAGE, followed by a high acrylamide
gel for the second-dimensional SDS-PAGE.
We demonstrate here that the combination of these three
techniques provides important supplementary information
on the synaptic vesicle proteome. We report the allocation of
proteins with highly purified synaptic vesicles from rat brain
that were previously not attributed to these organelles. Fur-
thermore, we identify several novel synaptic vesicle-asso-
ciated proteins with unknown function.
2 Materials and methods
2.1 Sample preparation
Isolation of synaptic vesicles was performed as previously
described [10]. In brief, for each gel electrophoretic analysis
three adult rats were anesthetized, and the entire brains were
rapidly removed and homogenized in preparation buffer
(5 mM Tris-HCl, 320 mM sucrose, pH 7.4). The homogenate
was centrifuged for 10 min at 10006g. The supernatant was
collected and the pellet was resuspended in preparation buf-
fer, and recentrifuged. Both supernatants were pooled and
the final pellet was discarded. Discontinuous Percoll gra-
dients were prepared by layering 7.5 mL supernatant onto
three layers of 7.5 mL Percoll solution (3, 10, and 23% v/v in
320 mM sucrose, 5 mM Tris-HCl, pH 7.4). After centrifuga-
tion for 7 min at 31 4006g, fractions containing synapto-
somes were collected, diluted in four volumes of preparation
buffer and centrifuged for 35 min at 20 0006g. For osmotic
lysis of synaptosomes, pellets were resuspended in 30 mL of
lysis buffer (5 mM Tris-HCl, pH 7.4). The suspension was
centrifuged for 1 h at 188 0006g, and the pellet was resus-
pended in 4 mL of sucrose gradient buffer (200 mM sucrose,
0.1 mM MgCl
2
, 0.5 mM EGTA, 10 mM HEPES-NaOH,
pH 7.4) and homogenized. The resulting suspension was
layered onto a continuous sucrose gradient ranging from 0.3
to 1.2 M sucrose (sucrose in 10 mM HEPES-NaOH, 0.5 mM
EGTA, pH 7.4) and centrifuged for 2 h at 85 0006g. From
top to bottom of the gradient 36 fractions (1 mL each) were
collected. In order to analyze the enrichment or dilution of
individual proteins during the purification procedure, sam-
ples were collected from each fractionation step.
2.2 Immunoisolation
The immunoisolation was performed as previously descri-
bed [10]. Magnetic beads (Dynabeads M-280, Dynal, Ham-
burg, Germany) were washed with TBS and incubated for
30 min in TBS containing 2% glycine, 2% lysine, and
0.5% saponin w/v, followed by blocking (twice for 10 min)
with TBS containing 2% lysine and 2% glycine w/v, and
washing for 10 min in TBS. Monoclonal anti-synaptic vesicle
protein 2 (SV2) antibody (3 mg) was coupled to 10
7
magnetic
beads by incubation for 1 h. For immunoisolation controls,
beads were coupled with nonspecific anti-mouse immuno-
globulins. Antibodies were crosslinked for 5 min with
0.1% glutaraldehyde in TBS. Crosslinking was stopped by
four washes with TBS containing 1% glycine and 1% lysine
w/v and two washes with TBS. Subsequently, 2% lysine and
2% glycine w/v (final concentration) were added to the
pooled vesicle fractions corresponding to sucrose concentra-
tions of 0.290.44 M. Pooled fractions were incubated with
the magnetic beads for 90 min at 47C. Beads containing the
bound vesicles were washed three times with TBS. Stripping
was performed by incubation for 2 min in 1 M Tris (pH 7.3),
and for additional 15 min in 0.1 M Na
2
CO
3
(pH 11) on ice.
Beads were washed twice with TBS before elution was per-
formed. For 1-D SDS-PAGE, proteins were eluted with
150 mL of 56 sample buffer (10% SDS, 0.5 M DTT,
5% glycerol, 0.006% Bromophenol blue, 400 mM Tris,
pH 6.8) over night at 377C. For dSDS-PAGE, elution was
performed with 150 mL of dSDS buffer (4% SDS, 2% mer-
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6252 J. Burr et al. Proteomics 2006, 6, 62506262
captoethanol, 10% glycerol, 0.017% Serva Blue G, 50 mM
Tris-HCl, pH 7.0) over night at 377C. For BAC/SDS-PAGE,
2% NP 40 in TBS (pH 7.4) was applied for 2 h on ice to elute
proteins. Samples for BAC/SDS-PAGE were subsequently
precipitated using 50% acetone.
2.3 1-D SDS-PAGE
Samples (250 mg) were incubated for 10 min at 407C before
loading onto 15% acrylamide SDS-gels (14 cm separation
length). Electrophoresis was performed as previously de-
scribed [12]. The gels were subjected to CBB staining.
2.4 dSDS-PAGE
Samples (250 mg) were incubated for 10 min at 407C before
loading onto 9% acrylamide tricine SDS-gels (14 cm separa-
tion length). Electrophoresis and equilibration was per-
formed according to Rais et al. [14]. A 15% acrylamide
Laemmli SDS-gel with double salt concentration in the buf-
fer was used for the second dimension [12]. The gels were
stained using CBB.
2.5 BAC/SDS-PAGE
Samples (250 mg) were resuspended in 20 mL of BAC sample
buffer and incubated at 607C for 5 min before loading onto
8% acrylamide BAC-gels (8 cm separation length). Electro-
phoresis and fixation were performed as previously de-
scribed [13]. The gel was subjected to CBB staining. For
separation in the second dimension, the equilibrated lane
was placed on top of a 15% acrylamide SDS-gel. Electropho-
resis was performed according to Laemmli [12]. The gels
were subjected to CBB staining.
2.6 In-gel digestion
All gels were subjected to manual gel cutting using a razor
blade for the 1-D gel or a shortened pipette tip for the 2-D
gels. For achieving the highest possible resolution, the 1-D
gel was cut into 238 thin slices of equal size. Excision of
polypeptides from the BAC/SDS-gel and dSDS-gel resulted
in 161 and 117 samples, respectively. Dissected gel pieces
were subjected to in-gel digestion protocols [15] which were
adapted for use on a Microlab Star digestion robot (Bonaduz,
Switzerland) [16]. Samples were reduced, alkylated, and
subsequently digested using bovine trypsin (sequencing
grade, Roche, Mannheim, Germany). The extracts were dried
in a vacuum centrifuge and stored at 2207C.
2.7 MALDI-TOF-MS
MALDI-TOF-MS experiments were performed on either a
Voyager-DE

STR (Applied Biosystems, Foster City, CA,

USA) or an Ultraflex TOF/TOF mass spectrometer (Bruker
Daltonics, Billerica, MA, USA). The samples were dissolved
in 5 mL of water/ACN/TFA (29:70:1 v/v/v). CHCA (3 mg/
mL; Bruker Daltonics) in water/ACN/TFA (50:50:0.5 v/v/v)
was used as the matrix. Analyte and matrix were spotted
consecutively in a 1:1 ratio on a stainless steel target and
dried under ambient conditions. Prior to analysis the dried
sample was washed with ice-cold 5% formic acid to reduce
salt contamination.
Spectra were externally calibrated with a Sequazyme

Peptide Mass Standards kit (Applied Biosystems) and

internally calibrated on a tryptic auto digestion peptide
(m/z 2163.06).
All spectra acquired on a Voyager-DE

STR were pro-

cessed using the Data Explorer version 4.3 (Applied Biosys-
tems), including noise filtering (correlation factor 0.7) and
advanced baseline correction (peak width 32, flexibility 0.5,
degree 0.1). Deisotoped peaks with an S/N of at least 5 were
used for database searching.
Spectra acquired on an Ultraflex TOF/TOF were pro-
cessed in flexAnalysis version 2.2 (Bruker Daltonics) using
the SNAP algorithm (S/N threshold: 3, maximal number of
peaks: 150, quality factor threshold: 80).
Proteins were identified using a MASCOTserver (Matrix
Science, Boston, MA, USA) (peptide mass tolerance: Ultra-
flex TOF/TOF 30 ppm; Voyager-DE STR 50 ppm, maximum
missed cleavages: 1) installed on a local server using the CDS
mammalian database (255 626 sequences, download date
21/01/2004, Celera Genomics, Rockville, MD, USA). Pro-
teins with a score of 67 or higher were considered significant
(p ,0.05). In the few cases where the score was lower than
67, the proteins have been included since they were identi-
fied by more than one gel electrophoretical method, by
Western blot analysis, or were obtained as protein mixtures.
All proteins listed in Supplementary Tables 2 and 3 (Supple-
mentary material) have been identified in biological dupli-
cates or triplicates.
2.8 Nano-LC ESI-MS/MS
Nano-LC separation was performed on a 1100 Nanoflow
Proteomics Solution (Agilent Technologies, Palo Alto, CA,
USA) using a PepMap

nanoscale C18 column (Dionex,

Sunnyvale, CA, USA). Water/ACN/formic acid (97:3:0.1 v/v/
v) was used as solvent A, and water/ACN/formic acid
(3:97:0.1 v/v/v) as solvent B. The samples were dissolved in
20 mL solvent A and 5 mL of the sample was injected and
trapped on a Zorbax 300 SB precolumn (0.3 mm65 mm,
5 mm; Agilent Technologies) with a flow rate of 20 mL/min.
Separation was performed on the nanoscale column using a
linear gradient (340% B for 30 min, 4080% B for 10 min,
80% B for 3 min, 803% B for 1 min, 3% B for 12 min) with
a flow rate of 200 nL/min. Mass spectrometric analysis was
performed online using a QTrap 2000 hybrid tandem mass
spectrometer (ABI MDS Sciex, Concord, ON). Data analysis
was undertaken by extracting the MS/MS data with the
MASCOT Daemon software (Matrix Science). Extracted
data were subjected to MASCOT (Matrix Science) data-
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Proteomics 2006, 6, 62506262 Animal Proteomics 6253
base search against the CDS mammalian database
(255 626 sequences) and a nonsense database (reversed
CDS mammalian database; 255 626 sequences) for the esti-
mation of false positive rates. Peptide mass tolerance was
set to 0.5 Da, MS/MS mass tolerance was set to 0.3 Da, and
maximum number of missed cleavages was set to 1. Pep-
tides yielding an ion score of 37 or higher were considered
significant (p ,0.05). At least two peptides were mandatory
for significant protein identification (i.e., protein score of 67
or higher). All proteins listed in Supplementary Table 1
(Supplementary material) were identified in biological
duplicates. Proteins listed in Supplementary Table 4 (Sup-
plementary material) were identified in several bands of one
biological sample.
2.9 Western blot analysis
SDS and BAC/SDS gels were blotted onto NC membranes
using a semidry apparatus. Blocking and washing was per-
formed as previously described [17]. For immunodetection,
ECL was applied. Primary antibodies were obtained from
Synaptic Systems (Gttingen, Germany) and Santa Cruz
(CA, USA). Secondary antibodies were obtained from Dako-
Cytomation (Glostrup, Denmark) for mouse antibodies and
Sigma (St. Louis, MO, USA) for rabbit antibodies, and both
were used at a dilution of 1:1000.
3 Results
3.1 Specificity of the immunoisolation protocol
The immunoisolation protocol using an mAb against a sur-
face-located epitope of the synaptic vesicle membrane pro-
tein SV2, yielded synaptic vesicles (approximately 100 mg per
rat brain) of high purity [10].
The specificity of the immunoisolation procedure was
evaluated using Western blot analyses to monitor abundance
changes during the vesicle isolation protocol. Equal amounts
of proteins were loaded to evaluate a potential enrichment or
reduction in marker proteins (Fig. 1). Immunopurification
of synaptic vesicles resulted in a strong enrichment of the
integral synaptic vesicle proteins SV2, synaptophysin,
synaptotagmin I, synaptogyrin, synaptoporin, vesicle-asso-
ciated membrane protein 2 (VAMP-2), secretory carrier-
associated membrane protein 1 (SCAMP 1), the vesicular
Figure 1. Western blot analysis monitoring the enrichment or reduction of proteins in fractions obtained during the synaptic vesicle iso-
lation procedure. 1, total brain homogenate; 2, crude synaptosomal fraction; 3, pure synaptosomal fraction; 4, osmotically lysed synapto-
somes; 5, supernatant after pelleting lysed synaptosomes; 6, crude synaptic vesicle fraction; 7, synaptic vesicle fractions 511 fromsucrose
gradient; 8, immunopurified synaptic vesicles. Protein (2 mg) was loaded per lane. The dilution of the primary antibodies were 1:1000 for
SV2, synaptophysin, synaptotagmin I, synaptogyrin, synaptoporin, VAMP-2, SCAMP 1, vGaT, vGluT1, vAChT, synapsin I, synapsin II,
b-tubulin, SNAP-25, syntaxin-1, munc18, NSF, ab-SNAP, clathrin heavy chain, rab5, Erk-1/2, GFAP, Calnexin; 1:500 for rab3A, rabphilin3A,
complexins 1 and 2, dynamin-1, Na,K-ATPase subunit alpha 5, CAT1, AMPA receptor subunits 2 and 3, PSD-93, PSD-95, peroxisomal
membrane protein of 70 kDa (PMP70); dilution for munc13 antibody 1:200, GAPDH 1:250, pyruvate kinase 1:250, Cav2.2 1:100, SAP102
1:400, F
0
F
1
-ATPase subunit a 1:10000. The molecular weights are indicated at the left margin.
2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com
6254 J. Burr et al. Proteomics 2006, 6, 62506262
neurotransmitter transporters vesicular GABA transporter
(vGaT), vesicular glutamate transporter (vGluT1), and vesic-
ular acetylcholine transporter (vAChT). Peripherally asso-
ciated proteins behaved differently. Whereas the amounts of
synapsin I and raphilin3A (rab3A) were slightly decreased
after immunoisolation, rab3A, synapsin II, and b-tubulin
were dramatically reduced or no longer immunodetected in
this fraction. Proteins involved in SNARE complex forma-
tion and/or exo- and endocytosis, including synaptosome-
associated protein of 25 kDa (SNAP-25), syntaxin-1, com-
plexins 1 and 2, munc13 and 18, N-ethyl maleimide sensitive
factor (NSF), ab-soluble NSFattachment protein (ab-SNAP),
the heavy chain of clathrin, dynamin-1, and rab5 were also
diminished in the immunoprecipitate. Only traces of the
glycolytic enzymes glyceraldehyde 3-phosphate dehy-
drogenase (GAPDH) and pyruvate kinase are present in the
immunoprecipitate, whereas extracellular signal-regulated
kinase (Erk)-1/2 can no longer be immunodetected after
release of synaptic vesicles from synaptosomes. Plasma
membrane localized proteins such as Na,K-ATPase, the
neuronal calcium channel Cav2.2, and calcium transporter 1
(CAT1) were already absent in the gradient fractions used for
immunoisolation. Accordingly, postsynaptic density (PSD)
proteins such as PSD-93, PSD-95, and synapse-associated
protein (SAP)102 were absent in these gradient fractions;
only traces of the a-amino-3-hydroxy-5-methyl-4-isoxazole
propionic acid (AMPA)-receptor were present. Putatively
contaminating proteins such as glial fibrillary acidic protein
(GFAP) and calnexin remain at a constant level indicating
nonspecific binding, whereas mitochondrial and peroxi-
somal markers were absent from the gradient fractions used
for immunoisolation. In addition, in control experiments,
nonspecific anti-mouse immunoglobulins G (IgGs) were
coupled to the immunobeads and immersed with the
synaptic vesicle fractions. This was followed by BAC/SDS-
PAGE and 2-D Western blot analysis. Also under these con-
ditions a strong immunosignal for GFAP was obtained (not
shown). This suggests that this protein binds nonspecifically
to the magnetic immunobeads and/or IgGs.
3.2 Protein separation by three gel-based techniques
For a comprehensive organellar proteome analysis, the pro-
teome of the immunoisolated synaptic vesicles was analyzed
using three different gel-based approaches in combination
with MS. 1-D SDS-PAGE was combined with nano-LC ESI-
MS/MS and the 2-D systems BAC/SDS-PAGE and dSDS-
PAGE were combined with MALDI-TOF-MS. All analyses
were performed as biological duplicates or triplicates.
Application of 1-DSDS-PAGE and CBB staining revealed
numerous protein bands ranging from approximately 10 to
300 kDa (Fig. 2A). The application of the 2-D BAC/SDS-
PAGE system revealed CBB stained polypeptides within the
same molecular mass range (Fig. 2B). Using the dSDS-
PAGE, the proteins in the high molecular mass range
(.200 kDa) were highly concentrated but not well separated
Figure 2. Separation of synaptic vesicle proteins by three differ-
ent gel systems. (A) 1-D SDS-PAGE, (B) 2-D BAC/SDS-PAGE, and
(C) 2-D dSDS-PAGE. For each separation, 150 mg of immunoiso-
lated synaptic vesicle protein was loaded. Polypeptides were
visualized by CBB staining. The distribution in the gel of several
previously identified synaptic vesicle proteins is indicated by
numbers: 1, SV2; 2, synapsin I; 3, synaptotagmin I; 4, synapto-
physin; 5, rab3A. Note the migration of hydrophobic proteins
above the diagonal in dSDS-PAGE (C, ag). a, vATPase V
0
sub-
unit c; b, SCAMP 5; c, DKFZp566N034; d, vGaT; e, vGluT1; f, SV2;
g, vATPase V
0
subunit a.
(Fig. 2C). Both the 2-D separation techniques resulted in the
migration of proteins along the diagonal. Following the
BAC/SDS-PAGE, the proteins were more broadly distributed
(Fig. 2B). In contrast, in the dSDS-system, proteins migrate
more closely to the diagonal. Defined protein spots were
consistently identified above the diagonal (Fig. 2C, ag).
These proteins are highly hydrophobic and include the vAT-
Pase V
0
subunit c (a), SCAMP 5 (b), DKFZp566N034 (c),
vGaT (d), vGluT1 (e), SV2 (f), and the vATPase V
0
sub-
unit a (g).
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Proteomics 2006, 6, 62506262 Animal Proteomics 6255
3.3 High yield of protein identification
Using the three different gel separation techniques, we
identified a total of 185 proteins in each of the duplicate or
triplicate gel analyses (Table 1, detailed information is given
in Supplementary Tables 13). Fifty-four proteins were iden-
tified in individual 1-D SDS-PAGE analyses in several pro-
tein bands (Supplementary Table 4). No false positive pro-
teins were observed using the same criteria for protein iden-
tification (two peptides yielding a score higher than 37). In
case of the 2-D gel techniques that allowed the excision of
visible spots, only 4% of the spots escaped protein identifi-
cation. Out of the 185 proteins, 143 proteins were identified
by 1-D SDS-PAGE. BAC/SDS-PAGE and dSDS-PAGE
resulted in the identification of 59 and 96 proteins, respec-
tively (Fig. 3). Only 35 proteins were identified by all three
separation techniques. Comparing the different gel tech-
niques, the overlap in the number of identified proteins was
22 for 1-D SDS-PAGE and dSDS-PAGE, 14 for 1-D SDS-
PAGE and BAC/SDS-PAGE, and 7 for dSDS-PAGE and
BAC/SDS-PAGE. Seventy-two proteins were identified only
by 1-D SDS-PAGE, and 3 and 32 proteins, respectively, were
identified exclusively by BAC/SDS-PAGE and dSDS-PAGE.
3.4 Wide range of protein functions
Identified proteins were grouped into several categories
according to their presumed subcellular localization and
function (Fig. 4). About half of the proteins were identified as
known synaptic vesicle proteins that either represented inte-
gral synaptic vesicle proteins, or proteins peripherally or
transiently associated with synaptic vesicles, including pro-
teins involved in endocytosis. The identified synaptic vesicle
proteins were grouped into transporters, SNAREs, vacuolar
proton pump subunits, synapsins, rab proteins, rab-inter-
acting proteins, and proteins modulating synaptic vesicle
exo- and endocytosis. Moreover, several cytoskeletal proteins
were identified (10%). The latter include actin, tubulin, neu-
rofilament proteins, spectrins, internexin, and proteins
involved in cytoskeletal structure and rearrangement as well
as organelle movement. Numerous guanine nucleotide tri-
phosphate (GTP)-binding proteins (6%) involved in various
signaling cascades were detected. All three experimental
approaches also identified chaperones (4%) and a large
number of metabolic enzymes (10%) involved in, e.g.,
glycolysis and lipid metabolism. Putative contaminating
proteins included mitochondrial proteins (2%) and 8% of the
proteins were derived from additional cellular sources.
Another 6% of the proteins have previously been allocated to
the presynaptic plasma membrane. Most interestingly, 4% of
the identified proteins have not previously been assigned to
the synaptic vesicle compartment.
3.5 Resolution of integral membrane proteins
Integral membrane proteins were detected by the three
technical approaches to a different extent. The relative con-
tribution of identified integral membrane proteins, soluble
proteins, and membrane-anchored proteins is shown in
Fig. 5. About one quarter (24%) of all proteins identified
Figure 3. Contribution of proteins identified by the three different
separation procedures. Out of the total of 185 proteins, 35 pro-
teins were identified by all three technical approaches. Over-
lapping identification: 22 for SDS-PAGE/dSDS-PAGE, 14 for SDS-
PAGE/BAC/SDS-PAGE, and 7 for dSDS-PAGE/BAC/SDS-PAGE.
Seventy-two proteins were identified solely by SDS-PAGE, 32 by
dSDS-PAGE, and three by BAC/SDS-PAGE.
Figure 4. Diagram of the per-
centage of proteins attributed to
presumed function and/or local-
ization. Proteins were grouped
into the following categories:
integral synaptic vesicle pro-
teins, vesicle-associated pro-
teins, endocytosis, cytoskeleton,
G-proteins, chaperones, meta-
bolic enzymes, plasma mem-
brane, mitochondria, presumed
contaminants, and novel pro-
teins.
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6256 J. Burr et al. Proteomics 2006, 6, 62506262
Table 1. Proteins identified by 1-D SDS-PAGE, BAC/SDS-PAGE, and dSDS-PAGE
Protein Accession numbers/
Database source
Gel system
Integral synaptic vesicle proteins
SV2 spt)Q02563 1-D SDS BAC/SDS dSDS
SV2B trm)Q63564 1-D SDS dSDS
Synaptogyrin 1 spt)Q62876 1-D SDS dSDS
Synaptogyrin 3 rf)XP_220220.2 1-D SDS
Synaptophysin spt)P07825 1-D SDS BAC/SDS dSDS
Synaptotagmin I spt)P21579 1-D SDS BAC/SDS dSDS
Synaptotagmin II rf)NP_796376.2 1-D SDS BAC/SDS
Synaptotagmin V spt)P47861 1-D SDS
Synaptotagmin XII spt)P97610 1-D SDS dSDS
Syntaxin 1 gb)AAB22526.1 1-D SDS dSDS
Syntaxin 1B2 rf)NP_036832.1 BAC/SDS dSDS
VAMP-1 trm)Q8CH14 1-D SDS dSDS
VAMP-2 rf)NP_036795.1 1-D SDS BAC/SDS
VAMP-B spt)Q9Z269 dSDS
vATPase V
0
subunit a1 spt)P25286 1-D SDS BAC/SDS dSDS
vATPase V
0
subunit c rf)NP_570836.1 dSDS
vATPase V
0
subunit d rf)XP_214672.2 1-D SDS BAC/SDS dSDS
vATPase V
1
subunit A1 rf)XP_340988.1 1-D SDS BAC/SDS dSDS
vATPase V
1
subunit B2 rf)NP_476561.1 1-D SDS BAC/SDS dSDS
vATPase V
1
subunit C rf)XP_216940.2 1-D SDS BAC/SDS
vATPase V
1
subunit D rf)XP_216742.1 1-D SDS BAC/SDS dSDS
vATPase V
1
subunit E1 rf)XP_216251.1 1-D SDS BAC/SDS dSDS
vATPase V
1
subunit F spt)P50408 1-D SDS dSDS
vATPase V
1
subunit G2 trm)Q8R2H0 BAC/SDS dSDS
vATPase V
1
subunit H trm)Q8BVE3 1-D SDS dSDS
vGaT spt)O35458 1-D SDS dSDS
vGluT1 trm)Q62634 1-D SDS dSDS
vGluT2 trm)Q9JI12 1-D SDS
ZnT-3 rf)XP_345643.1 1-D SDS dSDS
SCAMP 1 spt)P56603 1-D SDS dSDS
SCAMP 3 rf)XP_342280.1 1-D SDS dSDS
SCAMP 5 trm)Q9JKD3 1-D SDS dSDS
Reticulon 1 spt)Q64548 1-D SDS dSDS
Reticulon 3b trm)Q8VBU0 1-D SDS
Reticulon 4 spt)Q9JK11 1-D SDS dSDS
NTT4 gb)AAB24776.1 1-D SDS
Vat1 protein rf)XP_213484.2 1-D SDS
Vesicle-associated proteins
CaMKII alpha spt)P11275 1-D SDS
Cysteine string protein rf)NP_077075.1 1-D SDS
rab1 trm)Q7TPK7 1-D SDS
rab1A spt)P05711 dSDS
rab1B rf)XP_229035.1 1-D SDS BAC/SDS dSDS
rab2A spt)P05712 1-D SDS BAC/SDS
rab2B rf)XP_223991.1 dSDS
rab3A sp)P05713 1-D SDS BAC/SDS dSDS
rab3B spt)Q63941 1-D SDS BAC/SDS
rab3C dbj)BAA11302.1 1-D SDS BAC/SDS dSDS
rab4A gb)AAH62016.1 1-D SDS
rab5 trm)O88565 1-D SDS
rab5B rf)XP_213824.2 1-D SDS
rab5C rf)XP_213463.1 1-D SDS dSDS
rab6 rf)XP_344926.1 1-D SDS
rab6B rf)XP_343460.1 BAC/SDS
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Proteomics 2006, 6, 62506262 Animal Proteomics 6257
Table 1. Continued
Protein Accession numbers/
Database source
Gel system
rab7 pir)S01934 1-D SDS
rab8 trm)Q8K3X5 1-D SDS
rab10 pir)B42148 1-D SDS
rab11A rf)NP_112414.1 1-D SDS BAC/SDS
rab11B trm)Q9ET14 1-D SDS BAC/SDS
rab14 pir)E42148 1-D SDS BAC/SDS dSDS
rab16 pir)G42148 1-D SDS
rab18 rf)XP_225453.1 1-D SDS BAC/SDS
rab30 rf)XP_218916.1 dSDS
Rabconnectin-3b trm)Q9ERH3 1-D SDS
Rabphilin-3A spt)P47709 1-D SDS BAC/SDS
Septin 2 trm)Q91Y81 1-D SDS
Septin 5 rf)XP_359275.1 1-D SDS
Septin 7 spt)Q9WVC0 1-D SDS
Septin 11 rf)XP_223227.2 1-D SDS
b-SNAP rf)XP_345448.1 dSDS
SNAP-25 rf)NP_112253.1 1-D SDS
Synapsin I spt)P09951 1-D SDS BAC/SDS dSDS
Synapsin Ib gb)AAA42148.1 BAC/SDS dSDS
Synapsin II spt)Q63537 1-D SDS BAC/SDS dSDS
Synapsin IIb pir)D30411 dSDS
Syntaxin-binding protein 1 gb)AAA96350.1 1-D SDS BAC/SDS
Thy1 antigen prf)0902236A 1-D SDS dSDS
Endocytosis
AP2 alpha 1 spt)P17426 1-D SDS BAC/SDS dSDS
AP2 alpha 2 gb)AAH58099.1 1-D SDS BAC/SDS dSDS
AP2 beta 1 rf)XP_214419.2 1-D SDS BAC/SDS dSDS
AP2 mu rf)NP_446289.1 1-D SDS BAC/SDS
AP2-associated kinase rf)XP_232172.2 1-D SDS
Clathrin assembly protein AP180 pir)S36326 1-D SDS dSDS
Clathrin coat assembly protein AP17 rf)XP_346535.1 dSDS
Clathrin heavy chain spt)P11442 1-D SDS BAC/SDS dSDS
Dynamin-1 spt)P21575 1-D SDS BAC/SDS dSDS
EH-domain containing 1 rf)XP_215154.2 dSDS
NSF trm)Q9QUL6 1-D SDS BAC/SDS dSDS
Synaptojanin 1 trm)Q8CHC4 1-D SDS
Syntaxin 7 spt)O70257 1-D SDS
Syntaxin 12 trm)O88385 1-D SDS
Cytoskeleton
Actin alpha rf)XP_215801.2 1-D SDS
Actin beta rf)NP_112406.1 1-D SDS BAC/SDS dSDS
ARP2/3 subunit 2 rf)XP_217432.2 dSDS
ARP2/3 subunit 4 rf)XP_238365.2 dSDS
Capping protein alpha 2 rf)XP_347257.1 1-D SDS
EF1 alpha 1 rf)XP_227080.2 1-D SDS
EF1 alpha 2 sp)P27706 1-D SDS
Alpha-Internexin spt)P23565 1-D SDS BAC/SDS dSDS
MAL2A trm)Q7TPB7 1-D SDS
Myosin Va spt)Q9QYF3 1-D SDS
NF triplet M protein spt)P12839 1-D SDS BAC/SDS dSDS
Spectrin alpha 2 rf)NP_741984.2 1-D SDS
Spectrin beta 1 rf)XP_240072.2 1-D SDS
E-STOP protein trm)O88748 dSDS
Suppressor of profilin/p41 of ARP2/3 trm)Q99PD4 1-D SDS
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6258 J. Burr et al. Proteomics 2006, 6, 62506262
Table 1. Continued
Protein Accession numbers/
Database source
Gel system
Tubulin alpha prf)0812252A 1-D SDS BAC/SDS dSDS
Tubulin beta 2 gb)AAH60597.1 BAC/SDS dSDS
Tubulin beta 5 rf)NP_775125.1 1-D SDS
Tubulin beta 15 rf)XP_346523.1 BAC/SDS dSDS
G-proteins
ARF-like 10B trm)Q8VEH3 1-D SDS
ARF-like 10C trm)Q9CQW2 1-D SDS
Atlastin spt)Q8WXF7 1-D SDS
GAP1 like spt)Q9Z268 dSDS
Guanine nucleotide-binding protein G(O), alpha 2 sp)P30033 1-D SDS
Guanine nucleotide-binding protein G(O), beta 1 rf)XP_213170.2 1-D SDS
Ras-related C3 botulinum toxin substrate 1 (rac1) trm)Q9D859 1-D SDS
v-ral simian leukemia viral oncogene homolog A (ral A) rf)NP_112355.1 1-D SDS dSDS
RAP2B rf)NP_596901.1 1-D SDS
rasGAP-activating-like protein 1 spt)Q9Z268 1-D SDS
Transforming protein p21 (K-Ras) spt)P46203 1-D SDS
Chaperones
Calnexin precursor spt)P35565 dSDS
CCT eta rf)XP_216180.1 1-D SDS
CCT delta (chaperonin delta) trm)Q7TPB1 1-D SDS
DnaJ (Hsp40) homolog, subfamily C, member 13 rf)XP_135146.4 1-D SDS
Heat shock cognate 71 kDa protein rf)XP_212758.2 1-D SDS
Heat shock protein (HSP) 8 rf)NP_077327.1 dSDS
HSP 90-beta (HSP 84) spt)P34058 dSDS
Metabolic enzymes
Aldose reductase-like protein trm)Q91W30 dSDS
Chromaffin granule ATPase II homolog rf)XP_223390.2 1-D SDS dSDS
2,3-cyclic nucleotide 3-phosphodiesterase trm)Q64575 1-D SDS BAC/SDS dSDS
Cyclin-dependent kinase 6 rf)XP_342639.1 dSDS
Dihydrolipoamide acetyltransferase dbj)BAA01504.1 1-D SDS
Dipeptidyl aminopeptidase-related protein gb)AAC42062.1 1-D SDS
Fructose bisphosphate aldolase A spt)P05065 1-D SDS BAC/SDS dSDS
GAPDH trm)Q9QWU4 1-D SDS BAC/SDS dSDS
Glutamine synthetase spt)P09606 1-D SDS BAC/SDS dSDS
Lactate dehydrogenase A spt)P06151 1-D SDS BAC/SDS dSDS
Lactate dehydrogenase B rf)XP_129164.2 1-D SDS
MAPK1 rf)NP_446294.1 BAC/SDS dSDS
Monoglyceride lipase trm)Q8R431 1-D SDS dSDS
6-Phosphofructokinase, type C spt)P47860 1-D SDS
Protein kinase C alpha pir)KIMSCA 1-D SDS
Protein kinase C beta 1 pir)KIRTC1 1-D SDS
Potential phospholipid-transporting ATPase IA spt)P70704 1-D SDS
Pyruvate kinase 3 spt)P11981 1-D SDS BAC/SDS dSDS
Novel proteins
Da1-10 trm)Q7TP09 dSDS
Hypothetical protein rf)XP_342338.1 dSDS
Hypothetical protein DKFZp566N034 rf)XP_341115.1 1-D SDS dSDS
KIAA0587 protein rf)XP_230038.2 dSDS
Leucine zipper domain protein rf)XP_213213.2 dSDS
RIKEN cDNA 1200015A19 trm)Q9DBS2 1-D SDS BAC/SDS dSDS
Testis-specific adriamycin sensitivity protein rf)XP_216158.2 1-D SDS
WD repeat and FYVE domain containing 1 isoform 1 rf)XP_237323.2 1-D SDS
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Proteomics 2006, 6, 62506262 Animal Proteomics 6259
Table 1. Continued
Protein Accession numbers/
Database source
Gel system
Putative contaminating proteins
Plasma membrane
Contactin precursor spt)Q63198 dSDS
Glycoprotein m6a trm)Q812E9 1-D SDS
LSAMP spt)Q62813 1-D SDS
Myelin basic protein spt)P02688 1-D SDS BAC/SDS dSDS
Na,K-ATPase alpha 3 spt)P06687 1-D SDS dSDS
Na,K-ATPase beta 1 spt)P07340 1-D SDS BAC/SDS
NCAM140 spt)P13596 1-D SDS
Proteolipid protein rf)NP_112252.1 1-D SDS
VILIP-1 rf)NP_036818.1 1-D SDS
VILIP-3 rf)NP_059052.1 1-D SDS
Mitochondrial proteins
Aspartate aminotransferase, mitochondrial precursor spt)P00507 1-D SDS BAC/SDS
Creatine kinase, ubiquitous mitochondrial precursor spt)P30275 1-D SDS BAC/SDS dSDS
F
1
-ATPase A chain pdb)1MAB_A 1-D SDS
F
1
-ATPase B chain pdb)1MAB_B 1-D SDS
Others
Brain acid soluble protein 1 spt)Q05175 dSDS
GFAP trm)Q9Z2S0 BAC/SDS
Interleukin enhancer binding factor 2 rf)XP_227385.2 dSDS
Hemoglobin alpha 2 spt)P01966 1-D SDS
Hemoglobin beta 2 prf)0408174A 1-D SDS
Matrin 3 rf)XP_212889.2 BAC/SDS dSDS
NADPH-cytochrome P450 reductase spt)P00388 dSDS
PSD protein trm)Q9QX19 dSDS
Proteasome 26S non-ATPase subunit 3 rf)XP_213452.2 dSDS
Proteasome 26S non-ATPase subunit 8 rf)XP_214888.2 dSDS
Ribosomal protein S3 pir)R3RT3 dSDS
Ribosomal protein S10 rf)NP_112371.1 dSDS
Ribosomal protein S13 rf)XP_345331.1 dSDS
Ribosomal protein S16 rf)XP_341816.1 dSDS
Spindlin-like protein 2 (SPIN-2) rf)XP_346091.1 dSDS
The table provides the protein names, accession numbers, and the techniques applied for identification.
were integral membrane proteins, 38% were soluble, and
38% were attached to the membrane via a lipid anchor.
The relative distribution of proteins identified by 1-D
SDS-PAGE analysis was close to the total, 29% of the
proteins were integral membrane proteins, 28% were
soluble proteins, and 43% membrane-anchored proteins.
For BAC/SDS-PAGE, the distribution differed especially
regarding the contribution of membrane proteins (19%).
The majority of identified proteins was membrane-
anchored (42%). Soluble proteins contributed 39%.
dSDS-PAGE provided intermediate results, 25% of the
identified proteins were integral membrane proteins,
48% were soluble, and 27% were membrane-anchored
proteins.
To determine the efficiency of each technique for the
identification of hydrophobic proteins, we performed a com-
parative analysis of the grand average of hydrophobicity
index (gravy) scores (Fig. 6). The majority of proteins was
hydrophilic and revealed a gravy score between 0.1 and 20.6.
Some of the very hydrophilic proteins with gravy scores of
less than 20.6 were identified by all three techniques.
Hydrophobic proteins revealed gravy scores up to 1.1. BAC/
SDS-PAGE identified only one protein with a gravy score
higher than 0.1 (ZnT-3, 0.169). In contrast, 1-D SDS-PAGE
resulted in the identification of proteins with gravy scores up
to 0.9. Only dSDS-PAGE lead to the identification of the very
hydrophobic vATPase V
0
subunit c (gravy score 1.075) (com-
pare Fig. 1C, a).
2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com
6260 J. Burr et al. Proteomics 2006, 6, 62506262
Figure 5. Relative distribution of pro-
teins identified as integral membrane
proteins, cytosolic proteins and mem-
brane-anchored proteins. The four col-
umns represent all identified proteins
(total) and the proteins identified by the
three gel separation techniques.
Figure 6. Percentage distribu-
tion of identified proteins
according to hydrophobicity.
Gravy scores were grouped in
intervals of 0.1 for all three tech-
nical approaches (1-D SDS-
PAGE, BAC/SDS-PAGE, and
dSDS-PAGE).
4 Discussion
Our study reveals that the three gel electrophoretic tech-
niques applied are complementary for protein identification
and allow the identification of a large number of synaptic
vesicle proteins including novel protein candidates. About
19% of the 185 proteins were identified by all three methods.
The 1-D SDS-PAGE in combination with nano-LC ESI-MS/
MS was the most efficient technique for a comprehensive
proteome analysis. Yet, 23% of all proteins were identified by
the 2-D gel systems only. It, therefore, appears essential to
apply several complementary techniques for protein separa-
tion in order to obtain a comprehensive inventory of the
synaptic vesicle proteome.
Since conventional 2-DE employing IEF encounters dif-
ficulties in the separation of hydrophobic proteins, the 2-D
systems BAC/SDS-PAGE and dSDS-PAGE were especially
designed for the separation of membrane proteins [13, 14].
Interestingly, as compared to the two other techniques
applied, BAC/SDS-PAGE was less efficient for the identifi-
cation of hydrophobic proteins. The analysis of the gravy
score distribution provided additional information concern-
ing the efficiency of protein identification. Most of the iden-
tified proteins were hydrophilic and were equally identified
by all three systems. In contrast to BAC/SDS-PAGE, 1-D
SDS-PAGE, and dSDS-PAGE were most powerful in the
identification of hydrophobic proteins. It is noteworthy that
in dSDS-PAGE the migration of proteins with multiple
transmembrane domains differs from that of hydrophilic
proteins. When the separation is completed, hydrophobic
proteins are located above hydrophilic proteins. This migra-
tion behavior has recently been demonstrated by Rais et al.
[14]. Therefore, dSDS-PAGE allows a visual inspection of the
hydrophobicity of proteins.
A Western blot analysis of samples taken during the iso-
lation procedure revealed a specific enrichment of integral
synaptic vesicle proteins. This is in agreement with our pre-
vious electron-microscopical analysis using the same immu-
2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com
Proteomics 2006, 6, 62506262 Animal Proteomics 6261
noisolation protocol [10]. Although the t-SNAREs syntaxin-1
and SNAP-25 decrease in abundance in the sucrose gradient
fractions 511, they are still detectable in the immunoiso-
lated vesicle fraction. It has been reported that a portion of
these proteins resides on synaptic vesicles [18, 19]. Recently,
the mainly postsynaptically localized AMPA receptors have
also been reported to reside on presynaptic vesicles [20]. We
have reported earlier that marker proteins for ER, lysosomes,
Golgi, peroxisomes, mitochondria, nuclei, presynaptic, and
postsynaptic membranes are absent from immunoisolated
vesicles [10]. This suggests that the contaminating proteins
identified by MS bound nonspecifically to the immunobeads
or were detected as trace contaminants by the highly sensi-
tive mass spectrometric methods.
Many of the proteins have previously been identified as
integral or peripherally associated synaptic vesicle proteins
such as components of the vacuolar-type proton pump,
transport proteins involved in neurotransmitter uptake, and
trafficking proteins that participate in synaptic vesicle exo-
and endocytosis, and recycling [3]. These include the SNARE
proteins [21], the associated VAP proteins (VAMP-A and B)
[22] and members of the septin family [23], various transpor-
ter proteins including the vesicular amine transporter Vat1
[24], the vesicular glutamate [25] and GABA [26] transporters,
the zinc transporter 3 (ZnT-3) [27] or the Na
1
/Cl
2
-depend-
ent-like orphan transporter NTT4/Rxt1 [28], and three iso-
forms of the SCAMP family [29]. Other proteins of the
endomembrane system including members of the reticulon
family [30] and Yop1p (GP106 or polyposis locus protein 1
homolog) [31] have not previously been allocated to the
synaptic vesicle compartment. Moreover, numerous G-pro-
teins and metabolic enzymes were identified. G-proteins play
a fundamental role in cellular signaling and are presumably
involved in synaptic transmission [32]. The association of
metabolic enzymes, in particular, of the glycolytic pathway
with synaptic vesicles has been reported previously[10, 33].
The enzymes generate a high local ATP-concentration that
drives the neurotransmitter import into the synaptic vesicle
lumen [33]. Cytoskeletal proteins are involved in synaptic
vesicle transport [34], anchoring of the reserve pool [35], and
recycling of synaptic vesicles [36]. Chaperones have been
reported to interact with synaptic vesicles [37]. Since our
immunoisolation protocol using a monoclonal anti-SV2
antibody targets the entire synaptic vesicle pool of the brain,
we cannot differentiate between the subproteomes of vesicle
of defined neurotransmitter type. Probably, some of the pro-
teins are expressed only in subtypes of neurons whereas
others may have a ubiquitous distribution.
In addition, we identified a number of vesicle-associated
proteins of unknown function. Using the available sequence
information we performed an in silico analysis of the novel
proteins. In a number of cases, this allowed us to predict
similarities to conserved domains of known proteins. The
predicted protein WD repeat and FYVE domain containing
one isoform 1 contains three WD-40 repeats and is pre-
sumably soluble. The predicted protein testis specific adria-
mycin sensitivity protein is presumably soluble and might
function in the immune surveillance of the CNS. The pre-
dicted protein corresponding to RIKEN cDNA 1200015A19
is presumably soluble and reveals no significant similarities
to any protein in the databases. The hypothetical protein
DKFZp566N034 has six putative transmembrane domains
and reveals similarities to bacterial cation transporters. The
predicted protein corresponding to RIKEN cDNA
110031B06 (hypothetical protein LOC303183) reveals simi-
larity to SVAP1 and contains a t-SNARE domain as described
in SNAP-25 or SNAP-23 [38]. The hypothetical protein
rf)XP
2
342338.1 is related to ankyrin. The predicted protein
Dal-1 reveals no similarities to any of the proteins con-
tained in the databases with the exception of a coiled-coil
domain. KIAA0587 reveals similarities to the NCK-asso-
ciated protein 1 (Nap1). Nap1 interacts with rac and might
have a role in the suppression of apoptosis [39]. The pre-
dicted leucine zipper domain protein reveals no sequence
similarity to other proteins. Studies are underway to investi-
gate the regional distribution of the novel proteins and to
evaluate their functional role.
In summary, our analysis of immunoisolated synaptic
vesicle proteins using three different gel-based approaches in
combination with mass spectrometric methods resulted in
the identification of 185 proteins and thus permits a com-
prehensive analysis of the synaptic vesicle proteome. It
reveals that the number of membrane integral or vesicle-
associated proteins is considerably larger than previously
anticipated. The biochemical and functional characterization
of the newly identified constituents of the synaptic vesicle
proteome is expected to generate novel insight into synaptic
vesicle function and nerve terminal dynamics.
We thank Hamilton Robotics for providing the Microlab

Star digestion robot. This work was supported by the Deutsche

Forschungsgemeinschaft (SFB628, P16) (to W.V. and H.Z).
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