Analytical Biochemistry
journal homepage: www.elsevier.com/locate/yabio
a r t i c l e i n f o a b s t r a c t
Article history: The separation and detection of individual amyloid beta (Ab) aggregates by capillary electrophoresis with
Received 31 December 2011 laser-induced fluorescence detection (CE–LIF) was demonstrated. Samples were prepared with either Ab
Received in revised form 8 March 2012 (1–40) or Ab (1–42) peptides and were characterized by CE with ultraviolet (UV) absorbance detection
Accepted 14 March 2012
and transmission electron microscopy (TEM). Using thioflavin T (ThT) in the electrophoresis buffer, elec-
Available online 21 March 2012
trophoresis of aggregate-containing samples (5.0-s injection) produced up to several hundred narrow
(<20 ms FWHM [full width at half maximum]) fluorescence peaks. Injection of Ab (1–40) monomer sam-
Keywords:
ples resulted in no additional peaks compared with controls. The CE–LIF results were validated by bulk
Capillary electrophoresis
Fluorescence
ThT fluorescence measurements for the same samples. The potential of laser-induced fluorescence
Amyloid anisotropy (LIFA) with CE to characterize individual Ab aggregates also was investigated.
Fluorescence anisotropy Ó 2012 Elsevier Inc. All rights reserved.
Protein aggregation
Senile plaques composed of neurofibrillary tangles and poly- of time for the entire reaction landscape from monomer to fully
morphic amyloid beta (Ab)1 fibrils are hypothesized to be responsi- formed fibrils, where all forms of Ab would be distinguishable.
ble for the neurological degeneration associated with Alzheimer’s Current analytical techniques are far from this ideal. A variety
disease (AD) [1,2]. Aggressive research efforts have focused on devel- of techniques have been employed to study static Ab structures,
oping methods to dissociate or prevent the formation of amyloid mainly monomers or fully formed fibrils. These include imaging
fibrils [2,3]. Effective development of such treatments requires a techniques such as transmission electron microscopy (TEM),
thorough understanding of the formation and composition of Ab pla- atomic force microscopy, and total internal reflection fluorescence
ques. Ab (1–40) and Ab (1–42) peptides readily form amyloid fibrils microscopy [4,5]. In addition, spectroscopic and spectrometric
in vitro. The aggregation reaction from soluble monomeric peptide techniques such as circular dichroism, thioflavin T (ThT) and Con-
to insoluble mature fibrils is a complex multistep process that in- go red fluorescence, fluorescence correlation spectroscopy, light
cludes heterogeneous populations of dynamic polymorphic interme- scattering, surface plasmon resonance, nuclear magnetic reso-
diates [2]. These intermediates have been implicated to be cytotoxic; nance, and mass spectrometry have been applied to study Ab
however, the identity and structure of the cytotoxic species remain structures [6–12].
unknown. These multiple potential therapeutic targets make devel- Detecting and distinguishing monomeric Ab peptides, various
oping a treatment for AD extremely challenging. intermediate oligomeric aggregates, and fully formed fibrils within
An ideal technique to analyze Ab peptide aggregation would a sample mixture using one technique is an important challenge
provide concentration and structural information as a function that has not yet been met. Most of the aforementioned techniques
can detect larger peptide aggregates or fibrils, but they are incapa-
ble of simultaneously detecting smaller aggregated peptide struc-
⇑ Corresponding author. Fax: +1 225 578 3465.
tures. Imaging techniques, for example, have a size threshold
E-mail address: sdgilman@lsu.edu (S.D. Gilman).
1
Abbreviations used: Ab, amyloid beta; AD, Alzheimer’s disease; TEM, transmission
below which the peptide is not detectable. This size threshold also
electron microscopy; ThT, thioflavin T; SEC, size exclusion chromatography; CE, exists for light scattering detection. ThT fluorescence measure-
capillary electrophoresis; LIF, laser-induced fluorescence; LIFA, laser-induced fluo- ments allow the detection of peptide aggregates that have folded
rescence anisotropy; Tris, tris(hydroxymethyl) aminomethane; MO, mesityl oxide; into a b-sheet conformation that is more pronounced as a function
TFA, trifluoroacetic acid; HFIP, hexafluoroisopropanol; PBS, phosphate-buffered
of increasing aggregate size. The signal measured by bulk fluores-
saline; HPLC, high-performance liquid chromatography; UV, ultraviolet; PMT,
photomultiplier tube; FWHM, full width at half maximum. cence is also dominated by larger Ab aggregates that mask the
0003-2697/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.ab.2012.03.006
Analysis of Ab aggregates by CE–LIF / R.A. Picou et al. / Anal. Biochem. 425 (2012) 104–112 105
signal produced from smaller aggregates. Larger aggregates also 334 were prepared at 50 nM in Tris buffer. ThT was purchased
dominate bulk light scattering measurements. Although there is from Sigma (St. Louis, MO, USA). A stock solution of 289 lM ThT
no one technique to monitor all forms of Ab, all of the current tech- solution was prepared in Tris buffer, and working solutions of
niques are very informative and important within their limitations. ThT were prepared at 15.0 lM in Tris buffer.
Characterizing the structural pathway from monomeric pep-
tides to fibrillar aggregates involves measuring and characterizing Ab peptide sample preparation and characterization
the intermediate dynamic oligomeric structures for which little
information is available. Separations of Ab aggregates based on size Ab (1–40) peptide was purchased from the W.M. Keck Founda-
have been performed using several techniques, including ultracen- tion Biotechnology Research Laboratory at Yale University (New
trifugation, sedimentation velocity analysis, gel electrophoresis, Haven, CT, USA), and Ab (1–42) peptide was purchased from rPep-
size exclusion chromatography (SEC), and capillary electrophoresis tide (Bogart, GA, USA). Four types of Ab sample were studied in this
(CE) [13–23]. Although CE was first applied to the analysis of Ab work: Ab (1–40) monomer, Ab (1–40) fibril, Ab (1–42) monomer,
monomer in 1993 by Sweeney and coworkers [20], only a few re- and Ab (1–42) fibril. All Ab samples were prepared as described
ports regarding the application of CE to Ab analysis have been pub- previously [27,35]. Ab peptides were treated with trifluoroacetic
lished [13,21–27]. CE offers fast separations with higher peak acid (TFA) and hexafluoroisopropanol (HFIP) to remove any preex-
capacities and lower mass detection limits relative to other separa- isting aggregates. For the Ab monomer sample, the TFA and HFIP
tion methods applied to study Ab aggregation. Rapid separations were evaporated off and the peptide was dissolved in 10.00 mM
are especially important to minimize the disruption of various Tris at pH 7.79. For the Ab (1–40) fibril sample, the TFA and HFIP
aggregate structures when they are physically separated from the were evaporated off and the peptides were dissolved stepwise in
aggregate mixture. Another advantage of CE relative to gel electro- equal volumes of 2.0 mM NaOH and 2 phosphate-buffered saline
phoresis and SEC is that it does not require a gel or stationary (PBS) containing 22.8 mM phosphate, 274 mM NaCl, 5.4 mM KCl,
phase that can disrupt or otherwise alter aggregates due to shear and 0.1% NaN3 at pH 7.4. The samples were centrifuged at
and adsorption. Clodfelter and coworkers demonstrated that CE 50,000g for a minimum of 10 h at 4 °C. The supernatant was incu-
is a more gentle technique relative to SEC for separating protein bated at 37 °C for 7 days until there was no indication of further
aggregates of another aggregate-forming peptide, C8GLIP [28]. aggregation based on ThT fluorescence and high-performance li-
Laser-induced fluorescence (LIF) is a common on-column detec- quid chromatography (HPLC) analysis of monomer concentration.
tion technique for CE, and CE–LIF has been used to study Ab pep- The Ab (1–42) monomer and fibril samples were prepared using
tides [23]. Laser-induced fluorescence anisotropy (LIFA) is a less the same procedure with the following exceptions: the Ab (1–42)
frequently used detection method for CE, and CE–LIFA has been monomer sample was centrifuged for 30 min at 20,000g and 4 °C,
applied to study protein–protein and protein–nucleic acid interac- and the Ab (1–42) fibril sample was incubated for 2 days to form
tions [29–32]. Fluorescence anisotropy is a fluorescence phenome- mature fibrils. These changes were due to the faster aggregation
non that is dependent on the size of the fluorescent entity being kinetics of the Ab (1–42) peptide compared with that of the Ab
investigated. Sabaté and Saupe demonstrated the use of bulk ThT (1–40) peptide.
anisotropy to monitor aggregation kinetics of the fungal prion pro- Fibril growth was monitored using ThT fluorescence and TEM as
tein HET-s(218–289) [33]. Allsop and coworkers demonstrated described previously [12,27]. Depletion of Ab monomer during
that bulk time-resolved fluorescence anisotropy can be used for fibril formation was monitored using a Shimadzu HPLC–UV (ultra-
monitoring early stages of Ab aggregation using a fluorescein-la- violet) instrument with detection at 215 nm as described
beled Ab peptide [34]. CE with LIFA detection has the potential to previously [26,27,35]. Samples were characterized by CE–UV. The
be developed into a powerful tool for studying Ab aggregation. CE–UV analysis was performed using a Beckman Coulter P/ACE
CE is capable of resolving different Ab aggregate species, and LIFA MDQ equipped with a diode array detector, and the absorbance
could provide a sensitive on-line detection method that also pro- was monitored at 190 nm as described previously [27]. The capil-
vides information about the sizes of the separated species. To the lary (i.d. = 50 lm, o.d. = 366 lm) used for CE–UV was cut to
best of our knowledge, there are no reports of the application of 63.0 cm total length with a window created 53.0 cm from the inlet
CE–LIFA to study protein aggregation. end using The Window Maker (MicroSolv Technology, Eatontown,
The goal of this work was to separate and detect individual Ab NJ, USA). The Ab samples were vortexed prior to every injection to
aggregates labeled with ThT using CE with LIF detection. Further- resuspend any aggregates that settled to the bottom of the sample
more, the potential of LIFA to characterize individual Ab aggregates vial.
labeled with ThT was explored. Prior to CE analysis, fibril samples were buffer exchanged from
PBS to 10.00 mM Tris electrophoresis buffer at pH 7.79 [6]. This
Materials and methods step eliminated conductivity and composition differences between
the sample buffer (PBS, higher conductivity) and the electrophore-
Chemicals sis buffer (Tris, lower conductivity). The PBS buffer was used for
aggregation to form fibrils because this buffer is broadly used for
All solutions were prepared in 18 MX water obtained from a studies of Ab peptide fibril formation. The higher conductivity of
Modulab water purification system (U.S. Filter, Palm Desert, CA, PBS would result in high electrophoretic current and poor results
USA) unless otherwise noted. Tris(hydroxymethyl) aminomethane due to excessive Joule heating if it were used for CE. The mono-
(Tris) and methanol (99.8%) were purchased from Fisher Scientific mer-equivalent concentrations of all samples were determined to
(Fair Lawn, NJ, USA). Tris buffer was prepared at 10.00 mM, and the be 5 to 30 lM by HPLC–UV as described previously [35]. For CE
pH was adjusted to 7.79 with HCl. This Tris buffer was used for all analysis, the total sample volume was 50.0 ll, and it was placed
experiments unless otherwise noted. The buffer was filtered in a 200-ll Thermowell polypropylene tube (Corning, Corning,
through a 0.02-lm filter (Whatman, Hillsboro, OR, USA). Mesityl NY, USA).
oxide (MO) was purchased from Alfa Aesar (Ward Hill, MA, USA),
and working solutions of MO were prepared in Tris buffer at a con- CE with LIFA detection
centration of 0.2% (v/v). Coumarin 334 was purchased from Acros
Organics (Morris Plains, NJ, USA), and a stock solution was pre- The Ab samples were first characterized by CE–UV and then
pared at 1.00 lM in methanol. Working solutions of coumarin analyzed with the CE–LIFA system on the same day. The samples
106 Analysis of Ab aggregates by CE–LIF / R.A. Picou et al. / Anal. Biochem. 425 (2012) 104–112
were not transferred to different vials when switching from the RC filters and collected by a National Instrument PCI-6024E DAQ
CE–UV instrument to the CE–LIFA instrument. A laboratory-con- board at a scan rate of 1000 Hz. The CE–LIFA data collection was
structed instrument was used for all CE–LIFA experiments. The controlled by a program written with LabView (version 5.0), and
CE part of the instrument was similar to an instrument described the data were analyzed using Origin Pro 7.5, Synaptosoft MiniAnal-
previously [36]. ysis Program (version 6.0.7), and Microsoft Excel 2007.
CE was performed in an open tubular capillary (i.d. = 50 lm, Eq. (1) was used to calculate the fluorescence anisotropy, r,
o.d. = 366 lm) cut to 60.0 cm total length. A detection window where Ik and I\ are the measured parallel and perpendicular fluo-
was created at 23.0 cm from the inlet end. A new capillary was ini- rescence intensities relative to the polarization of the excitation
tially conditioned with a manual syringe pump as follows: 400 ll source. The factor G corrects the anisotropy for the differences in
of 1.00 M NaOH, 400 ll of 18 MX water, and 2 400 ll of Tris buf- sensitivities of the two PMTs. For this work, G was empirically ad-
fer. The total rinse time was approximately 60 min. The capillary justed to 1 by optimizing the potentials applied to both PMTs so
was rinsed twice between different sample types with 400 ll of that the responses and sensitivities were equal for a small fluores-
Tris electrophoresis buffer using a manual syringe pump. The fluo- cent molecule, Lucifer Yellow (Molecular Probes, Eugene, OR, USA),
rescent neutral marker (coumarin 334) was injected for 1.0 s at from 250 nM to 1 lM (data not shown):
25.0 kV prior to the Ab sample injection for 5.0 s at 25.0 kV. This Ik GI?
injection order minimized potential interactions between Ab and r¼ : ð1Þ
Ik þ 2GI?
the neutral marker because all forms of Ab detected migrated
slower than the neutral marker. The Ab samples were vortexed
prior to every injection. The electrophoretic potential was applied Microplate reader fluorescence
using a Spellman CZE1000R high-voltage power supply (Hauppa-
uge, NY, USA) at 25.0 kV (417 V/cm). The electrophoretic current All Ab samples were also analyzed by bulk ThT fluorescence
was 5 to 6 lA. This system was not temperature controlled, unlike using a BMG Lab Technologies FLUOstar 430 microplate reader
the commercial CE–UV system. (Offenbur, Germany). The instrument and data acquisition were
Fig. 1 presents a schematic of the detection system for the controlled by FLUOstar software (version 3.02-0). Here, 40 ll of
CE–LIFA instrument. The light source was a 445-nm diode laser each sample was transferred from the 200-ll polypropylene tubes
(model LDCU12/7532, Power Technology, Alexander, AR, USA). used for CE to separate wells of 96-well Corning 3650 black non-
The laser produced a continuous beam of polarized radiation, and transparent microplates. Consistent with the CE experiments, ThT
the measured power was 30.0 mW. The beam was attenuated with was added at 15 lM to each sample well. The excitation was set
a neutral density filter to 3.0 mW and focused onto the capillary to 440 ± 12 nm with a bandpass filter, and the emission was col-
window with a plano-convex lens (focal length = 25.4 mm). The lected at 485 ± 12 with a bandpass filter.
fluorescence emission was collected with a 20 microscope objec-
tive lens (numerical aperture = 0.4) at 90° relative to the excitation Results and discussion
beam. The emission was directed through a notch filter
(441.6 ± 10 nm, MK Photonics, Albuquerque, NM, USA) to remove The primary goal of this work was to separate and detect indi-
Rayleigh scattered light from the laser and a bandpass filter vidual Ab peptide aggregates by CE–LIF using ThT. ThT is a com-
(490 ± 10 nm, Omega Optical, Brattleboro, VT, USA) to selectively mon fluorescence probe used to study Ab aggregation because it
detect amyloid-bound ThT fluorescence. After being spectrally fil- is selective for amyloid aggregates, and its excitation/emission
tered, the emission was spatially filtered with a 1000-lm pinhole. maxima shift from 330/440 to 440/490 nm when it noncovalently
The emission was then separated into its parallel and perpendicu- binds to amyloid aggregates [11,12,37]. ThT was added to the elec-
lar polarized components (relative to the excitation source) with a trophoresis running buffer to bind to the Ab aggregates on-column
broadband polarizing cube beamsplitter (10FC16PB.3, Newport, [23]. This approach eliminates the potential for ThT to alter the Ab
Irvine, CA, USA). Both emission components were detected simul- aggregates’ sizes and shapes during the aggregation process while
taneously and equidistant from the capillary by two identical pho- still being used as an effective fluorescent label for their on-column
tomultiplier tubes (PMTs, H9306-04, Hamamatsu, Bridgewater, NJ, detection. At the excitation and emission wavelengths used in this
USA). The PMT collecting parallel emissions (PMTk) was at 1000 V, work, fluorescence interference from unbound ThT is minimal [11].
whereas the PMT collecting perpendicular emissions (PMT\) was The concentration of ThT (15 lM) was chosen based on previous
at 935 V. The outputs of the PMTs were filtered by 500-Hz lowpass publications that used 10 and 20 lM ThT [11,26] for Ab aggregate
detection. This is above the critical micelle concentration of free
ThT reported by Khurana and coworkers (3.8 ± 0.5 lM) [38].
k The work presented is distinct from the previous study by Kato
and coworkers [23] because the instrument used here was de-
signed to detect peaks resulting from individual aggregated Ab
j i
species, requiring the use of sufficiently fast data sampling and
electronic filters [39,40]. In addition, the ThT fluorescence mea-
h sured after CE separation was validated by measuring bulk ThT
g
fluorescence for the same samples.
f
e Detection of individual aggregates by CE–LIF
Figs. 3–6 for clarity. The two-channel data are discussed later (see
‘‘Fluorescence anisotropy of individual Ab aggregates’’ section).
Four different Ab sample types were prepared and used for this
work: Ab (1–40) monomer, Ab (1–40) fibril, Ab (1–42) monomer,
and Ab (1–42) fibril. In nature, Ab (1–42) and Ab (1–42) are pro-
duced by cleavage of the same parent peptide at different sites.
Both Ab (1–40) and Ab (1–42) are known to be present in amyloid
plaques, and Ab (1–42) has been shown to aggregate much more
rapidly in vitro and is difficult to prepare in a purely monomeric
form [1,19,27]. Both peptides will aggregate to form amyloid fibrils
that fluoresce on ThT binding and are similar in appearance in TEM
images [1,13,19,27]. The two fibril samples were prepared to con-
tain large mature fibrils that would bind to ThT, resulting in
enhanced fluorescence. The Ab (1–40) monomer and Ab (1–42)
monomer samples were prepared to be free of aggregates and
serve as negative controls. A representative TEM of each sample
type is shown in Fig. 2. The Ab fibril samples (Fig. 2B and D) show
clear evidence of fibrillar aggregates. The Ab monomer samples do
not show fibrillar aggregates by TEM (Fig. 2A and C); therefore,
these samples were not expected to bind to ThT and cause ThT Fig.3. Electropherograms with LIF detection for Ab (1–40) monomer without ThT
fluorescence enhancement [11,12,37]. (A) and with 15 lM ThT (B) in the electrophoresis buffer (10.00 mM Tris at pH 7.79).
Fig. 3A and B show electropherograms with LIF detection for the The peak at 59 s in each electropherogram is the neutral marker (NM), coumarin
Ab (1–40) monomer sample without ThT and with ThT in the run- 334. Coumarin 334 was injected for 1.0 s at 25.0 kV prior to the Ab sample injection
for 5.0 s at 25.0 kV. The separation potential was 25.0 kV (417 V/cm). The dip in the
ning buffer, respectively. As expected, no peaks were observed in
baseline at 47 s in panel B is due to a ThT vacancy zone.
the absence of ThT (Fig. 3A). In contrast, in the presence of ThT, a
few small sharp peaks were detected for Ab monomer (Fig. 3B). ThT fluorescence similar to amyloid fibrils. We are currently
The migration times of these peaks appear to be random for consec- investigating ThT fluorescence in the presence of synthetic nano-
utive injections, and approximately the same numbers of peaks particles due to their potential to produce false positives in amyloid
were observed in electropherograms of the electrophoresis buffer fibril assays. The Ab (1–40) monomer sample also was analyzed by a
with ThT for which no Ab was injected (data not shown). These CE–UV method previously developed by Picou and coworkers [26].
peaks are hypothesized to be a result of unfiltered particulate A representative CE–UV electropherogram of the Ab (1–40) mono-
matter (non-Ab particles) in the running buffer that can enhance mer sample is available in the Supplementary material (Fig. S1). This
Fig.2. TEM images of Ab (1–40) monomer (A), Ab (1–40) fibril (B), Ab (1–42) monomer (C), and Ab (1–42) fibril (D). Samples were buffer exchanged into 10.00 mM Tris buffer
at pH 7.79 prior to imaging (scale bar = 500 nm).
108 Analysis of Ab aggregates by CE–LIF / R.A. Picou et al. / Anal. Biochem. 425 (2012) 104–112
The presence of large Ab aggregates in the Ab (1–40) fibril sam- with ThT without ThT
ple was confirmed by CE–UV, TEM, and bulk ThT fluorescence mea- Ab (1–40) monomer 10 ± 3 1±1
surements. In the CE–UV electropherogram (Fig. 4C), the broad Ab (1–40) fibril 99 ± 23 13 ± 4
peak at 313 s and the sharp peaks from 300 to 400 s are typical Ab (1–42) monomer 176 ± 27 5±2
Ab (1–42) fibril 300 ± 46 300 ± 30
for Ab samples containing mature fibrils [27]. Bulk ThT fluores-
cence was higher for the Ab (1–40) fibril sample compared with Note: Peaks detected are averages with standard deviations from three injections.
the Ab (1–40) monomer sample (Table 2), consistent with the All peaks with signal/noise ratios greater than 3 were included.
Analysis of Ab aggregates by CE–LIF / R.A. Picou et al. / Anal. Biochem. 425 (2012) 104–112 109
Note: All data are presented as averages with standard deviations from three injections or wells. Data are normalized to the values for the Ab (1–42) fibril sample. The CE
values were calculated by summing the integration of the peaks detected in the electropherograms.
110 Analysis of Ab aggregates by CE–LIF / R.A. Picou et al. / Anal. Biochem. 425 (2012) 104–112
In the presence of ThT, a broad peak (FWHM = 9 s) at 98 s that Fig. 6A and B present electropherograms of Ab (1–42) fibrils
was separated from most of the peaks attributed to individual without ThT and with ThT in the running buffer, respectively.
aggregates (Fig. 5B) was obtained for the Ab (1–42) monomer sam- Fig. 6A shows that analysis of the Ab (1–42) fibrils resulted in many
ple. Analysis of the Ab (1–40) fibril sample by CE–LIF with ThT also sharp peaks in the absence of ThT that are thought to be due to scat-
resulted in a broad peak that comigrated with most of the peaks tered light from individual aggregates, similar to the scattering ob-
attributed to individual aggregates (Fig. 4B). These broad peaks served for the Ab (1–40) fibril sample (Fig. 4A). Table 1 shows that an
in Figs. 4 and 5B could be due to oligomeric structures, which are average of 300 peaks were detected for the Ab (1–42) fibril sample in
too small to produce large distinct peaks for individual aggregates the absence of ThT, and the peaks were much larger than those ob-
but are large enough to bind ThT and enhance its fluorescence. served for Ab (1–40) fibrils. This is consistent with the microplate
Biancalana and Koide discussed minimal b-sheet requirements measurements for the samples without ThT (Table 2). The large peak
for binding and fluorescence enhancement of ThT using a heights for the Ab (1–42) fibrils relative to the Ab (1–40) fibrils,
peptide self-assembly mimic [37], but it is not certain exactly what without ThT, suggest that the Ab (1–42) fibrils are larger.
Ab aggregates (size or shape) meet the minimum b-sheet Adding ThT to the running buffer resulted in an increase in the
requirements. intensity of the peaks detected for the Ab (1–42) fibril sample.
Ab (1–42) fibrils also were analyzed by CE–LIF (Fig. 6) and CE– Most of the peaks were off-scale initially, and a neutral density
UV. The CE–UV electropherograms (Fig. S2 in Supplementary mate- (ND) filter (10.7% transmission) was used to attenuate the emission
rial), bulk ThT fluorescence (Table 2), and TEM data (Fig. 2D) from for the electropherogram shown in Fig. 6B. The inset of 6B high-
Ab (1–42) fibrils clearly indicate that large aggregates were present lights a few of the Ab (1–42) aggregate peaks, showing that most
in the sample. Unlike the Ab (1–40) fibrils (Fig. 4), the CE–UV data peaks are well resolved from each other. The average values for to-
of the Ab (1–42) fibrils showed no signs of Ab monomer (Fig. S2). tal peaks presented in Table 1 are the same for the Ab (1–42) fibril
The absence of a monomer peak for Ab (1–42) fibrils is due to sample with and without ThT, but the peak intensities are much
the fast aggregation kinetics and the smaller monomer equilibrium higher with ThT in the running buffer. The total signal from the
concentration of the Ab (1–42) peptide relative to that of the Ab Ab (1–42) fibril sample increases by a factor of 8 when analyzed
(1–40) peptide [19]. in the presence of ThT (Table 2).
Fig.6. Electropherograms with LIF detection of Ab (1–42) fibrils analyzed without Fluorescence anisotropy of individual Ab aggregates
ThT (A) and with ThT (B) in the electrophoresis buffer. A neutral density filter was
used in panel B to attenuate the emission (10.7% transmission), and the y axis in
panel B was adjusted to account for this. The inset in panel B shows a 0.2-s segment
The results presented in the above two sections of Results and
of the electropherogram displaying several of the Ab (1–42) aggregate peaks. The discussion demonstrate that this CE–LIF method can separate Ab
experimental conditions for panels A and B are the same as in Fig. 3. aggregates, providing information about their numbers, electro-
Analysis of Ab aggregates by CE–LIF / R.A. Picou et al. / Anal. Biochem. 425 (2012) 104–112 111
with bulk ThT fluorescence measurements, CE–LIF provides a pro- [18] D.M. Hartley, D.M. Walsh, C.P. Ye, T. Diehl, S. Vasquez, P.M. Vassilev, D.B.
Teplow, D.J. Selkoe, Protofibrillar intermediates of amyloid b-protein induce
file of the aggregate population based on electrophoretic mobili-
acute electrophysiological changes and progressive neurotoxicity in cortical
ties, fluorescence intensities, and numbers of individual neurons, J. Neurosci. 19 (1999) 8876–8884.
aggregate peaks. The studies presented here focused on analysis [19] A. Jan, O. Gokce, R. Luthi-Carter, H.A. Lashuel, The ratio of monomeric to
of Ab aggregates, but they should be applicable to studies of other aggregated forms of Ab40 and Ab42 is an important determinant of amyloid-b
aggregation, fibrillogenesis, and toxicity, J. Biol. Chem. 283 (2008) 28176–
amyloidogenic peptides. 28189.
Initial studies of CE–LIFA to characterize individual Ab aggre- [20] P.J. Sweeney, J.G. Darker, W.A. Neville, J. Humphries, P. Camilleri,
gates yielded encouraging results, but significant improvements Electrophoretic techniques for the analysis of synthetic amyloid b-A4-related
peptides, Anal. Biochem. 212 (1993) 179–184.
must be made to apply this effectively to obtain relative size and [21] G. Thorsen, J. Bergquist, A. Westlind-Danielsson, B. Josefsson, Stereoselective
shape information for Ab aggregates. determination of amino acids in b-amyloid peptides and senile plaques, Anal.
Chem. 73 (2001) 2625–2631.
Acknowledgments [22] E. Varesio, S. Rudaz, K.-H. Krause, J.-L. Veuthey, Nanoscale liquid
chromatography and capillary electrophoresis coupled to electrospray mass
spectrometry for the detection of amyloid-b peptide related to Alzheimer’s
This work was supported by Louisiana State University, the Na- disease, J. Chromatogr. A 974 (2002) 135–142.
tional Science Foundation (CHE-0505972, S.D.G.), the Rosalinde [23] M. Kato, H. Kinoshita, M. Enokita, Y. Hori, T. Hashimoto, T. Iwatsubo, T.
Toyo’oka, Analytical method for b-amyloid fibrils using CE-laser induced
and Arthur Gilbert Foundation/AFAR Award (I.K.), and the Louisi-
fluorescence and its application to screening for inhibitors of b-amyloid
ana Board of Regents (I.K.). Ryan Picou was supported by a Louisi- protein aggregation, Anal. Chem. 79 (2007) 4887–4891.
ana Board of Regents Fellowship. Rebekah Cerqua was supported [24] A. Kishita, S. Nishino, T. Togashi, Y. Nishida, Formation of methionine sulfoxide
of amyloid b-peptide (1-40) by Cu(bdpe)/H2O2 system, Synth. React. Inorg.
by National Science Foundation Research Experiences for Under-
Met.–Org. Nano-Met. Chem. 35 (2005) 677–681.
graduates (REU, CHE-0648841). The authors thank the Louisiana [25] T. Frackowiak, T. Baczek, R. Kaliszan, B. Zbikowska, M. Glensk, I. Fecka, W.
State University School of Veterinary Medicine Microscopy Center Cisowski, Binding of an oxindole alkaloid from Uncaria tomentosa to amyloid
in the Department of Comparative Biomedical Sciences for TEM protein (Ab1–40), Z. Naturforsch. C 61 (2006) 821–826.
[26] R. Picou, J.P. Moses, A.D. Wellman, I. Kheterpal, S.D. Gilman, Analysis of
work. monomeric Ab (1–40) peptide by capillary electrophoresis, Analyst 135 (2010)
1631–1635.
[27] R.A. Picou, I. Kheterpal, A.D. Wellman, M. Minnamreddy, G. Ku, S.D. Gilman,
Appendix A. Supplementary data Analysis of Ab (1–40) and Ab (1–42) monomer and fibrils by capillary
electrophoresis, J. Chromatogr. B 879 (2011) 627–632.
Supplementary data associated with this article can be found, in [28] D.K. Clodfelter, M.A. Nussbaum, J. Reilly, Comparison of free solution capillary
electrophoresis and size exclusion chromatography for quantitating non-
the online version, at http://dx.doi.org/10.1016/j.ab.2012.03.006.
covalent aggregation of an acylated peptide, J. Pharm. Biomed. Anal. 19 (1999)
763–775.
References [29] Q.-H. Wan, X.C. Le, Studies of protein–DNA interactions by capillary
electrophoresis/laser-induced fluorescence polarization, Anal. Chem. 72
[1] D.M. Walsh, D.J. Selkoe, Deciphering the molecular basis of memory failure in (2000) 5583–5589.
Alzheimer’s disease, Neuron 44 (2004) 181–193. [30] R.J. Whelan, R.K. Sunahara, R.R. Neubig, R.T. Kennedy, Affinity assays using
[2] H. Levine III, Small molecule inhibitors of Ab assembly, Amyloid 14 (2007) fluorescence anisotropy with capillary electrophoresis separation, Anal. Chem.
185–197. 76 (2004) 7380–7386.
[3] T. Takahashi, H. Mihara, Peptide and protein mimetics inhibiting amyloid b- [31] P. Yang, R.J. Whelan, Y. Mao, A.W.-M. Lee, C. Carter-Su, R.T. Kennedy,
peptide aggregation, Acc. Chem. Res. 41 (2008) 1309–1318. Multiplexed detection of protein–peptide interaction and inhibition using
[4] T. Ban, Y. Goto, Direct observation of amyloid growth monitored by total capillary electrophoresis, Anal. Chem. 79 (2007) 1690–1695.
internal reflection fluorescence microscopy, Methods Enzymol. 413 (2006) 91– [32] L. Ye, X.C. Le, J.Z. Xing, M. Ma, R. Yatscoff, Competitive immunoassay for
102. cyclosporine using capillary electrophoresis with laser induced fluorescence
[5] C. Ha, C.B. Park, Ex situ atomic force microscopy analysis of b-amyloid self- polarization detection, J. Chromatogr. B 714 (1998) 59–67.
assembly and deposition on a synthetic template, Langmuir 22 (2006) 6977– [33] R. Sabaté, S.J. Saupe, Thioflavin T fluorescence anisotropy: an alternative
6985. technique for the study of amyloid aggregation, Biochem. Biophys. Res.
[6] I. Kheterpal, K.D. Cook, R. Wetzel, Hydrogen/deuterium exchange mass Commun. 360 (2007) 135–138.
spectrometry analysis of protein aggregates, Methods Enzymol. 413 (2006) [34] D. Allsop, L. Swanson, S. Moore, Y. Davies, A. York, O.M.A. El-Agnaf, I. Soutar,
140–166. Fluorescence anisotropy: a method for early detection of Alzheimer b-peptide
[7] A. Lomakin, D.B. Teplow, Quasielastic light scattering study of amyloid b- (Ab) aggregation, Biochem. Biophys. Res. Commun. 285 (2001)
protein fibril formation, Protein Pept. Lett. 13 (2006) 247–254. 58–63.
[8] J. Ryu, H.-A. Joung, M.-G. Kim, C.B. Park, Surface plasmon resonance analysis of [35] B. O’Nuallain, A.K. Thakur, A.D. Williams, A.M. Bhattacharyya, S. Chen, G.
Alzheimer’s b-amyloid aggregation on a solid surface. From monomers to Thiagarajan, R. Wetzel, Kinetics and thermodynamics of amyloid assembly
fully-grown fibrils, Anal. Chem. 80 (2008) 2400–2407. using a high-performance liquid chromatography-based sedimentation assay,
[9] S.A. Funke, E. Birkmann, F. Henke, P. Gortz, C. Lange-Asschenfeldt, D. Riesner, D. Methods Enzymol. 413 (2006) 34–74.
Willbold, Single particle detection of Ab aggregates associated with [36] K.F. Schrum, J.M. Lancaster III, S.E. Johnston, S.D. Gilman, Monitoring
Alzheimer’s disease, Biochem. Biophys. Res. Commun. 364 (2007) 902–907. electroosmotic flow by periodic photobleaching of a dilute, neutral
[10] R. Tycko, Characterization of amyloid structures at the molecular level by solid fluorophore, Anal. Chem. 72 (2000) 4317–4321.
state nuclear magnetic resonance spectroscopy, Methods Enzymol. 413 (2006) [37] M. Biancalana, S. Koide, Molecular mechanism of thioflavin-T binding to
103–122. amyloid fibrils, Biochim. Biophys. Acta 2010 (1804) 1405–1412.
[11] H. Levine III, Multiple ligand binding sites on Ab (1–40) fibrils, Amyloid 12 [38] R. Khurana, C. Coleman, C. Ionescu-Zanetti, S.A. Carter, V. Krishna, R.K. Grover,
(2005) 5–14. R. Roy, S. Singh, Mechanism of thioflavin T binding to amyloid fibrils, J. Struct.
[12] H. Naiki, F. Gejyo, Kinetic analysis of amyloid fibril formation, Methods Biol. 151 (2005) 229–238.
Enzymol. 309 (1999) 305–318. [39] Y.H. Rezenom, A.D. Wellman, L. Tilstra, C.D. Medley, S.D. Gilman, Separation
[13] S. Sabella, M. Quaglia, C. Lanni, M. Racchi, S. Govoni, G. Caccialanza, A. and detection of individual submicron particles by capillary electrophoresis
Calligaro, V. Bellotti, E. De Lorenzi, Capillary electrophoresis studies on the with laser-light-scattering detection, Analyst 132 (2007) 1215–1222.
aggregation process of b-amyloid 1–42 and 1–40 peptides, Electrophoresis 25 [40] H. Ahmadzadeh, R. Dua, A.D. Presley, E.A. Arriaga, Automated analysis of
(2004) 3186–3194. individual particles using a commercial capillary electrophoresis system, J.
[14] Y.-M. Kuo, M.R. Emmerling, C. Vigo-Pelfrey, T.C. Kasunic, J.B. Kirkpatrick, G.H. Chromatogr. A 1064 (2005) 107–114.
Murdoch, M.J. Ball, A.E. Roher, Water-soluble Ab (N-40, N-42) oligomers in [41] S.D. Gilman, A.G. Ewing, Analysis of single cells by capillary electrophoresis
normal and Alzheimer disease brains, J. Biol. Chem. 271 (1996) with on-column derivatization and laser-induced fluorescence detection, Anal.
4077–4081. Chem. 67 (1995) 58–64.
[15] Y.-F. Mok, J. Howlett Geoffrey, Sedimentation velocity analysis of amyloid [42] B. O’Nuallain, S. Shivaprasad, I. Kheterpal, R. Wetzel, Thermodynamics of Ab
oligomers and fibrils, Methods Enzymol. 413 (2006) 199–217. (1–40) amyloid fibril elongation, Biochemistry 44 (2005) 12709–12718.
[16] H.-W. Klafki, J. Wiltfang, M. Staufenbiel, Electrophoretic separation of bA4 [43] S.P. Radko, A. Chrambach, Separation and characterization of sub-lm- and
peptides (1–40) and (1–42), Anal. Biochem. 237 (1996) 24–29. lm-sized particles by capillary zone electrophoresis, Electrophoresis 23
[17] G. Bitan, Structural study of metastable amyloidogenic protein oligomers by (2002) 1957–1972.
photo-induced cross-linking of unmodified proteins, Methods Enzymol. 413 [44] J.R. Lakowicz, Principles of Fluorescence Spectroscopy, Kluwer Academic/
(2006) 217–236. Plenum, New York, 1999.