supplementary materials for...for anti-ttr antibody (rabbit anti-ttr, dako; pre-labeled with biotin)...
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Supplementary Materials for
Peptide probes detect misfolded transthyretin oligomers in plasma of
hereditary amyloidosis patients
Joseph D. Schonhoft, Cecilia Monteiro, Lars Plate, Yvonne S. Eisele, John M. Kelly,
Daniel Boland, Christopher G. Parker, Benjamin F. Cravatt, Sergio Teruya,
Stephen Helmke, Mathew Maurer, John Berk, Yoshiki Sekijima, Marta Novais,
Teresa Coelho, Evan T. Powers, Jeffery W. Kelly*
*Corresponding author. Email: [email protected]
Published 13 September 2017, Sci. Transl. Med. 9, eaam7621 (2017)
DOI: 10.1126/scitranslmed.aam7621
The PDF file includes:
Materials and Methods
Fig. S1. The B β-strand of TTR labels TTR50–127 oligomers.
Fig. S2. The minimal binding motif is VAVHVF.
Fig. S3. Determination of probe stoichiometry in MTTR oligomers.
Fig. S4. Additional microscopy images of salivary gland biopsies stained with the
B-peptide.
Fig. S5. Probe B-1 linearly labels MTTR oligomers in human healthy plasma.
Fig. S6. A similar probe B-1 structure-activity relationship is found when MTTR
oligomers are incubated in healthy plasma.
Fig. S7. Labeling of the high-MW SEC fraction in FAP patients by the B-peptide
is fluorophore-independent.
Fig. S8. Alanine substitutions on probe B-1 have identical effects on
incorporation of the peptide into the high-MW fraction of FAP V30M patient
plasma.
Fig. S9. B-1 is the only peptide of the TTR β-strands that incorporates into the
high-MW fraction of patient plasma.
Fig. S10. Diazirine-containing probe B-2 selectively labels oligomeric TTR.
Fig. S11. Schematic of probe B-2 non-native TTR gel quantification method and
representative data.
Fig. S12. Probe B-1 does not cross-react with the anti-TTR antibody (DAKO,
catalog no. A0002).
www.sciencetranslationalmedicine.org/cgi/content/full/9/407/eaam7621/DC1
Fig. S13. Correlation of spectral counts in the MS1 spectra of the diazirine-
containing B-2 targets from V30M FAP patients (average of three patients) with
plasma concentration.
Fig. S14. Validation of N-terminally cleaved non-native TTR as a target of the B-
peptide in V30M FAP patient plasma.
Other Supplementary Material for this manuscript includes the following:
(available at
www.sciencetranslationalmedicine.org/cgi/content/full/9/407/eaam7621/DC1)
Table S1. Full summary of MudPIT LC-MS/MS data presented in Fig. 5 (Excel
format).
Table S2. All raw data for experiments where n < 20 (Excel format).
Materials and Methods
Estimation of Non-Native TTR in Patient Plasma.
We estimate the concentration of non-native TTR in plasma to be in the low nanomolar
range. For this, the most useful data is the B-2 SEC/SDS PAGE assay used in Fig. 4, Fig. 6 and
Fig. 7. Use of the B-1 fluorescence SEC assay in Fig. 3 would be inappropriate as proteomics
experiments and rhodamine gel experiments confirm that this peptide differentially labels several
other proteins in the high MW fraction of patients and thus use of this data would result in
artificially high predicted concentrations.
In the B-2 assay, the probe is first added to patient plasma, photo-crosslinked,
fractionated by SEC and then rhodamine is ‘clicked’ onto proteins that are labeled in the high
MW SEC fraction (MW > ~200 kDa). Next the proteins are run in an SDS PAGE gel with a
standard curve of a protein labeled quantitatively with a single alkyne. This procedure is depicted
in Fig. S11. From this ‘alkyne’ standard curve we convert the rhodamine signal at the 13.5 kDa
TTR band to a concentration. For example, the average signal for the V30M FAP patients is 5.9
nM from the standard curve. After consideration of dilution by SEC and other manipulations
described in the methods, from peptide incubation to gel, there is a dilution factor of 6.25.
Therefore, on average ~37 nM (6.25 x 5.9) of B-2 peptide labeled TTR in the initial incubation
with plasma. Given stoichiometry’s ranging from 2:1 to 0.3:1 for the MTTR oligomers (B :
Oligomer) and the range of TTR labeling in FAP patients would result in a concentration range
of approximately 10-200 nM (based on monomer concentration). Further, considering that the
patient oligomers MW are ~0.5 MDa or greater by SEC (~10-100 monomers) would result in a
sub-nM oligomer concentration. We approach these numbers with a high degree of caution as
many assumptions are required.
C-terminal TTR fragment (TTR50-127) expression and purification: The protein coding
sequence was cloned into a plasmid upstream of an IPTG inducible T7 promoter (pMMHa). E.
coli BL21 containing the TTR50-127 plasmid were grown to an OD of 0.6 and induced at 37 C
with 1 mM IPTG and the protein was allowed to express overnight. After induction and protein
expression, the majority of the protein was found within inclusion bodies. The E. coli cells were
lysed by sonication and centrifuged at 15,000 x g. The supernatant was discarded and the
insoluble fraction was resuspended in 10 mL 6M guanidinium-HCl and sonicated for 35 minutes
in a water bath, followed by vigorous stirring for 2 hrs. The mixture was then centrifuged again
at 15,000 x g for 15 minutes. The supernatant was then dialyzed against 4 L of 25 mM Tris pH
8.8, followed by injection onto a Source15Q strong anion exchange column (GE healthcare). A
linear gradient was then run to elute the protein (Buffer A 25 mM Tris pH 8.8, 2 M Urea, Buffer
B: 25 mM Tris pH 8.8, 2 M Urea, 1 M NaCl). The fractions containing TTR50-127 were then
concentrated and injected onto a Sephadex 75 gel filtration column, run in 25 mM Tris pH 8.8, 2
M Urea. The purified protein was then dialyzed into 5 mM NH4HCO4, frozen by submersion in
liquid N2 and lyophilized.
Analysis of non-native recombinant oligomers / recombinant tetramers by Native PAGE
Formation of oligomers was monitored using Novex NativePAGE 4-16% Bis-Tris Gels
following the manufacturers protocol. After loading each sample into the gel, the gel was run for
100 min at 150 V and stained for total protein or imaged by fluorescence using a Biorad
ChemiDoc MP system.
Peptide screen: To quantify peptide incorporation into recombinant oligomers (TTR50-127
and MTTR), each candidate probe (2 M) was incubated overnight with 50 M oligomeric TTR
followed by injection onto an Agilent SEC-3 or SEC-5 column and monitored for fluorescence
(495 ex., 520 em.). A fluorescein standard curve was used to compare results from day to day.
The results represent the area of the fluorescently labeled high MW peak.
Peptide stoichiometry determination: we incubated 20 M of the B-1 peptide (the
identical concentration of probe used in analysis of the patient samples) with 50 M MTTR
oligomerized for different time periods, and then we separated the high MW labeled peak using
the same SEC method as above. The high MW fraction was denatured using guanidinium
chloride and the amount of B-1 was measured by the fluorescence intensity of the denatured
sample on an Aviv ATF 105 spectrofluorimeter at 25 °C, using an excitation wavelength of 490
nm and recording emission spectra between 600 and 500 nm. A standard curve with B-1 in the
same denaturing conditions was used to determine the amount of B-1 in the high MW fraction,
which was then divided by the amount of protein in that fraction. To determine quenching, an
emission spectrum was taken prior to denaturation and compared to a B-1 emission spectrum at
the determined concentration in the denaturation experiment. This procedure is depicted in fig.
S3.
Collection of plasma samples: Blood from healthy volunteers was obtained from The
Scripps Research Institute’s Normal Blood Donor Services Center. The blood was collected in
tubes with anticoagulant, followed by centrifugation at 1500 g for 20 min. The resulting
supernatant (plasma) was carefully removed and centrifuged for an additional 20 min to remove
any remaining cells. Aliquots of the clarified plasma were stored at −20°C. Blood from V30M
FAP patients (from Portugal and Japan), asymptomatic V30M mutation carriers (from Portugal)
and Portuguese and Japanese controls were also collected in anticoagulant coated tubes, and
plasma was prepared in the same way at the corresponding center. Remaining blood samples
(from WT cardiomyopathy patients and the non-V30M patients) were collected at Columbia
University College of Physicians and Surgeons (NY) and at the Boston Amyloidosis Center.
Plasma was transferred to 1.5 mL cryovials with a cap and stored at −75 °C until it was shipped.
After shipment in dry ice, frozen patient plasma samples were kept at −20°C. All plasma samples
were thawed on ice before being used. This study was approved by the respective Institutional
Review Boards in Portugal, Japan, NY, Boston and The Scripps Research Institute, and informed
consent was obtained from the participants.
Salivary gland biopsies staining with Thioflavin T, Anti-TTR antibody and B-1-Biotin:
Deparaffinization and re-hydration was performed according to the following protocol: 2 x 5 min
washes with 100% xylene, followed by 3 minutes wash with 50 : 50 xylene : ethanol, followed
by 3 minutes washes with ethanol 100%, 95%, 70%, 50%, and 2 x 3 min with water.
For Thioflavin T staining, slices were incubated for 10 minutes at room temperature with
1% aqueous Thioflavin-T (AnaSpec. Inc Ultra Pure Grade, Cat # 88306; filtered before each use
with a 0.22 µm syringe filter). Following incubation with Thioflavin T, slices were washed 2
times with 80% ethanol, and then covered with coverslip in Vectashield Mounting Medium.
Slices were allowed to dry overnight in the dark.
For anti-TTR antibody (rabbit anti-TTR, DAKO; pre-labeled with biotin) and B-1-Biotin
staining, slices were submitted to a mild permeabilization protocol as follows: 2 x 5 min washes
with cold methanol, followed by 2 x 5 min with PBS, 2 x 5 min with PBS-0.4% Triton X-100
(Fisher Scientific), and 20 min with 0.05% saponin (Acros Organics, Cat# 41923-1000) and 3 x
5 min wash with PBS. Slices were then incubated overnight at room temperature with antibody
(1:200 dilution), B-1-Biotin probe (100 M in DMSO) or DMSO (Vehicle). On the next day,
slices were washed 3 x 10 min with PBS, followed by 1 hour incubation with Streptavidin
Alexa®-594 Conjugated. Lastly, slices were washed 3 x 5 min with PBS, and covered with
coverslip Vectashield® Mounting Medium. Slices were allowed to dry overnight in the dark.
Probe B-2 non-native TTR gel quantification method: For sample sets in which we
directly compared non-native TTR labeling by in-gel rhodamine fluorescence in different patient
or control groups (asymptomatic V30M versus V30M FAP or Pre versus 12 mo tafamidis) we
used a standardized method. Plasma (47.5 L) was added to 2.5 L of 1mM probe B-2 and
rapidly mixed by pipetting up and down. The samples were then incubated overnight at 37C
(14-15 hours) and crosslinked in a 96-well plate as described above, run through a Bio-Spin® P-
30 column, followed by injection onto an SEC column (Agilent Bio SEC-3 4.6 x 300 mm, run at
1 mL/min in 10 mM sodium phosphate buffer pH 7.6, 100 mM KCl). The high MW peak
containing proteins that elute in the void volume and with a MW >200 kDa were collected (5-6
mL eluting peak). 200 L of this fraction was then taken and subjected to reaction with
rhodamine azide via the copper catalyzed azide-alkyne click reaction (1). After completion of the
click reaction, the proteins were precipitated with methanol/chloroform to remove any
unconjugated rhodamine dye. The resulting protein pellet was resuspended in 50 L of Laemmli
buffer containing DTT and boiled for 5 minutes. 20 L of the sample was then analyzed by SDS
PAGE (4-12% BOLT Gel, Invitrogen, run at 200 V for 30 minutes). To compare band intensities
between multiple gels, we also loaded in each gel a standard curve. We used a 28 kDa
recombinant protein (retroaldolase) labeled quantitatively at a single cysteine with an alkyne
using a maleimide derivative (retroaldolase-alkyne). Thus for each sample set, we separately
conjugated rhodamine azide from an identical fluorophore stock to retroaldolase-alkyne and
loaded varying concentrations of the rhodamine-labeled retroaldolase. This procedure is depicted
in fig. S11. All gels were imaged on a Biorad ChemiDoc MP system and bands were quantified
in ImageLab (Biorad).
Biotin pull-down experiments: samples were first subjected to the CuAAC click reaction
with N3-Diazo-Biotin. After biotin conjugation, each sample was precipitated with
methanol/chloroform and resuspended in 500 L of 8 M Urea, 1 X PBS, 5 mM EDTA, followed
by the addition of 140 L of 10% SDS. Following resuspension, the samples were then reduced
by adding TCEP (10 mM final concentration) and incubated for 30 min at room temperature.
The free sulfhydryls were then blocked by adding iodoacetamide to a final concentration of 12
mM and incubated at room temperature for 30 minutes in the dark. After reduction and
alkylation, the samples were then added to 6 mL of PBS containing 100 L of Streptavidin-
conjugated agarose beads and incubated at room temperature with gentle rocking overnight. The
biotin beads were then washed once with 1X PBS, 0.5% SDS, 5 mM EDTA, 3 times with 2 M
Urea, 1X PBS, 5 mM EDTA, and 3 times with 1X PBS. The samples were then eluted from the
beads by incubation with 100 L of dithionite containing solution (42 mM sodium dithionite,
0.5% SDS, 1X PBS, pH ~ 7) for 30 min at room temperature. The samples were then run in a
denaturing gel for western blot analysis or prepared for mass spectrometry analysis as described
below.
Anti-TTR western analysis: gels were transferred to nitrocellulose, blocked with Odyssey
blocking buffer (Licor), and probed with the anti-TTR antibody (rabbit anti-TTR, DAKO Inc.,
#A0002) or a C-terminal TTR specific antibody made by immunizing mice against TTR50-127 (a
gift from Westermark and colleagues). Membranes were then imaged using a Licor infared
imager following the addition of an anti-rabbit or anti-mouse IR-800 secondary antibody (Licor).
Fig. S1. The B β-strand of TTR labels TTR50–127 oligomers. (A) AFM image of TTR50-127
oligomers that were used to assay incorporation. (B) Quantification of incorporation of each
candidate TTR-derived peptide probe into TTR50-127 oligomers using size exclusion
chromatography. Bars represent mean of three different experiments; error bars represent s.d..
Fig. S2. The minimal binding motif is VAVHVF. Relative peptide incorporation as measured
by Native PAGE. Each probe B-1 deletion mutant labeled with fluorescein was incubated with 1-
week-old MTTR oligomers. After fluorescence imaging of the Native PAGE gel, the oligomer
band density was normalized to the probe B-1 sequence signal (AINVAVHVFR). Bars represent
mean of three different experiments; error bars represent s.d..
Fig. S3. Determination of probe stoichiometry in MTTR oligomers. A) Standard curve of
probe B-1 in 6 M Guanidine-HCl. B) Method for determination of fluorescence quenching and
stoichiometry of probe B-1 bound to recombinant oligomers. C) Representative emission scan
showing ~10 fold quenching of the fluorescein fluorescence intensity when probe B-1 is bound
to the MTTR oligomers.
Fig S4. Additional microscopy images of salivary gland biopsies stained with the B-peptide.
A) V30M FAP salivary gland biopsy images. B) Healthy control salivary gland biopsy images.
ThT: thioflavin T. Samples from FAP patients (n=2) and non-FAP controls were (n=2) were
used. Images shown represent samples from one patient and one control.
Fig. S5. Probe B-1 linearly labels MTTR oligomers in human healthy plasma. Increasing
concentrations of MTTR derived oligomers were incubated in healthy human plasma from three
different donors and next these oligomers were selectively labeled using probe B-1. The
concentration of probe B-1 in the high molecular weight SEC fraction increased linearly with the
increase in non-native TTR concentration (r2=0.9215; y=21.8x +35.05); all error bars represent
s.d.
0 2 4 6 8 10 12 140
50
100
150
200
250
300
350
400
450
[MTTR] in the high MW fraction (mM)
Inte
gra
ted H
igh
MW
Peak A
rea
Fig. S6. A similar probe B-1 structure-activity relationship is found when MTTR oligomers
are incubated in healthy plasma. MTTR oligomers were incubated in plasma of three healthy
donors and the incorporation of the probe B-1 alanine mutants was measured by size exclusion
chromatography (see Fig. 1F for similar results in buffer). Each bar represents the mean of the
three plasma samples for a specified mutant; all error bars represent s.d.
Inte
gra
ted H
igh M
W P
eak A
rea
I26
A
N2
7A
V2
8A
B-1
V3
0A
H3
1A
V3
2A
F3
3A
0
500
1000
1500
2000
2500
A25I26N27V28A29V30H31V32F33R34
Fig. S7. Labeling of the high-MW SEC fraction in FAP patients by the B-peptide is
fluorophore-independent. DisulfoCy5-B was incubated with V30M FAP patient samples (n=5)
and control samples (n=5) and the high MW SEC peak fluorescently labeled was quantified. Box
plot whiskers represent the 2.5–97.5% range. P value was calculated using unpaired two tailed t-
test.
Fig. S8. Alanine substitutions on probe B-1 have identical effects on incorporation of the
peptide into the high-MW fraction of FAP V30M patient plasma. The alanine mutants
incorporation was measured in three separate patient plasma samples and three healthy control
samples using the size exclusion chromatography method. For comparison, B-1 data from all
patients and controls analyzed with probe B-1 is shown (patients: n=45, controls: n=62). Each
bar represents the mean of the three plasma samples for a specified mutant; all error bars
represent s.d.. P values were calculated using unpaired two-tailed t-test (*** represents P <
0.0001, ** represents P < 0.01 and * represents P < 0.05).
I26
A
N2
7A
V2
8A
B-1
V3
0A
H3
1A
V3
2A
F3
3A
0
500
1000
1500
2000
Inte
gra
ted H
igh M
W P
eak A
rea
Healthy controls FAP patients
A25I26N27V28A29V30H31V32F33R34
ns
nsns
**
*
*ns
***
Fig. S9. B-1 is the only peptide of the TTR β-strands that incorporates into the high-MW
fraction of patient plasma. The 8 -strands were incubated with patient plasma (n=3 for strands
A, and C-H; n=45 for B-1) and peptide incorporation was measured by size exclusion
chromatography. Each bar represents the mean of the three plasma samples for a specified
mutant; all error bars represent s.d.
A B C D E F G H0
500
1000In
tegra
ted H
igh M
W P
eak A
rea
Fig. S10. Diazirine-containing probe B-2 selectively labels oligomeric TTR. A) Probe B-2
selectively labels non-native oligomeric TTR but not mutant or WT tetramers. B) Probe B-2
labels oligomeric TTR but not the folded monomer.
Fig. S11. Schematic of probe B-2 non-native TTR gel quantification method and
representative data. After incubation with probe B-2, the samples were photo-crosslinked,
separated by SEC using the Agilent Bio SEC-3 4.6 x 300 mm column and N3-Rhodamine was
then clicked onto the probe B-2 alkyne handle. The samples were then analyzed by reducing
SDS PAGE and the bands were quantified by densitometry and then normalized to the total
protein present. To compare samples between different gels, each gel was loaded with a standard
curve of retroaldolase labeled quantitatively with a single alkyne as described in the Methods.
The gel above depicts 5 plasma samples from V30M asymptomatic carriers (5 unique
individuals) and 5 V30M FAP patients (5 unique individuals).
Fig. S12. Probe B-1 does not cross-react with the anti-TTR antibody (DAKO, catalog no.
A0002). MTTR or probe B-1 was spotted onto the nitrocellulose membrane and either directly
imaged (fluorescein channel) or probed with the polyclonal Anti-TTR antibody from DAKO.
Fig. S13. Correlation of spectral counts in the MS1 spectra of the diazirine-containing B-2
targets from V30M FAP patients (average of three patients) with plasma concentration.
Approximate human plasma concentrations were obtained from the Plasma Proteome Database
(http://www.plasmaproteomedatabase.org, accessed October 1, 2016).
Fig. S14. Validation of N-terminally cleaved non-native TTR as a target of the B-peptide in
V30M FAP patient plasma. (A) N-terminally cleaved non-native TTR elutes in the high MW
fraction. SEC fractionation of patient #5 (representative example) with probe B-2 and visualized
by rhodamine conjugation to the alkyne handle (magenta box) or by anti-TTR western blot using
a C-terminal specific antibody (dark-green box). Patient sample numbers correspond to the same
patient sample labels that were shown in Fig. 6. (B) Western blot of plasma samples analyzed by
SDS PAGE and probed with a monoclonal C-terminal specific anti-TTR antibody. Patient
numbers correspond to Fig. 6 (C) N-terminally cleaved TTR is not generated by a protease
during the peptide incubation period. Identical results were observed with or without the addition
of a protease inhibitor cocktail (Roche Complete Protease Inhibitor Cocktail)