Supplementary Information

Quantitative proteomics of tau and Aβ in detergent

fractions from Alzheimer’s disease brains

Soumya Mukherjee,1†* Celine Dubois,1 Keyla Perez,1 Shiji Varghese,1 Ian E. Birchall,1
Miranda Leckey,1 Natalia Davydova,3 Catriona McLean,1,2 Rebecca M. Nisbet,1
Blaine R. Roberts1§, Qiao-Xin Li,1 Colin L. Masters,1 Victor A. Streltsov1*

1
The Florey Institute, The University of Melbourne, Parkville, Victoria 3010, Australia
2
Department of Anatomical Pathology, Alfred Hospital, Prahran, Victoria 3004, Australia
3
National Deuteration Facility, Australian Nuclear Science and Technology Organization, Lucas Heights,
NSW, Australia

†
Current Address: Biomolecular Mass Spectrometry and Proteomics, Utrecht University, Padualaan 8,
3584CH, Utrecht, Netherlands
§
Current Address: Department of Biochemistry, Department of Neurology, Emory University, School of
Medicine, 1510 Clifton Road, Rollins Research building G223, Atlanta, GA 30322 USA

*Corresponding Authors: s.mukherjee@uu.nl, vstreltsov@unimelb.edu.au

1
Supplementary Tables

Table S1. Demographics of AD and disease-control (DCN) ALS brain samples. NA, not
applicable.

ID Diagnosis Age PMI (hrs) Sex Braak

AD01 AD 66.6 9.5 M VI
AD02 AD 60.4 24 F VI
AD03 AD 78.8 9.5 M VI
AD04 AD 48 18 F VI
AD05 AD 67 31 M VI
AD06 AD 72.4 7.5 M VI
AD07 AD 87 29.5 F III
AD08 AD 48.2 28.5 F VI
AD09 AD 75.4 37.5 F V
AD10 AD 75.4 83 M VI
AD11 AD 72.5 19 M VI
DCN01 ALS 70.2 41 F NA
DCN02 ALS 43.7 31 F NA
DCN03 ALS 66.9 21 M NA
DCN04 ALS 62.4 48 F NA
DCN05 ALS 72.6 20.5 M NA
DCN06 ALS 76.6 39 M NA
DCN07 ALS 51.8 45 F NA
DCN08 ALS 70.2 34.5 F NA
DCN09 ALS 70.3 17 F NA
DCN10 ALS 61.5 47 F NA

2
Table S2. Antibodies used for immunoassays and immunolabeling EM.

Antibody Epitope Suppler

Mouse monoclonal primary antibodies
TAU1 Tau (1-22) In-house
RNF5 Tau (35-44) In-house
HJ8.5 Tau (25-30) Creative Biolabs (NS-090CN)
AT8 Tau (pSer202+pThr205) Thermo Fisher (MN1020)
WO2 Aβ (2-8) Sigma (MABN10)
6E10 Aβ (3-8) Abcam (AB49682)
1E8 Aβ (1-2) Sigma (MABN639)
6F/3D Aβ (9-14) Dako MO872
Rabbit polyclonal primary antibodies
K9JA Tau (243-441) Dako A0024(K9JA)
Ubiq Ubiquitin Sigma
Human monoclonal primary antibodies
Aducanumab Aβ (3-7) Biogen (BIIB037)
Secondary gold conjugated antibodies
Gold, 10 nm Goat anti-mouse IgG Sigma (G7652)
Gold, 5 nm Goat anti-rabbit IgG Sigma (G7277)
Gold, 6 nm Goat anti-human IgG Jackson ImmunoResearch Labs
Control antibody
IgG Anti-mouse IgG Sigma

3
Table S3. Tau and Aβ peptides absolute quantitation (fmol/mg brain) of tryptic and Lys-N
peptides, respectively, from the high-resolution MS data acquisition from the sarkosyl-insoluble
materials derived from the AD (n=8) brains.

Peptides AD01 AD02 AD04 AD05 AD06 AD08 AD09 AD10 Mean SD
tau24-44 11.1 18.9 18.9 6.2 33.5 0.9 8.1 1.4 12.4 11.0
N-term tau25-44 11.5 19.3 22.1 7.1 33.5 1.0 10.6 1.9 13.4 11.0
tau tau45-67 3.3 4.4 4.5 1.1 9.6 0.2 2.0 0.2 3.2 3.1
tau68-87 2.0 2.6 2.5 1.4 4.8 0.1 1.7 0.2 1.9 1.5
tau88-126 2.9 6.3 8.9 4.4 16.5 0.4 2.7 1.1 5.4 5.3
tau171-180 68.0 66.9 214.8 11.5 144.6 12.5 118.2 30.1 83.3 71.5
tau181-190 33.5 44.5 85.0 23.6 71.6 3.7 14.8 7.5 35.5 29.8
PRR tau181-194 34.2 99.8 339.7 94.5 40.9 8.7 29.1 16.5 82.9 109.1
tau tau195-209 2.7 6.7 6.3 3.9 8.9 0.3 2.0 0.6 3.9 3.1
tau210-224 0.5 2.0 2.0 2.0 3.2 1.3 1.9 1.0 1.7 0.8
tau212-221 25.9 7.6 57.6 26.9 6.1 2.9 9.8 6.7 17.9 18.4
tau212-224 32.2 81.3 73.3 33.9 90.0 3.3 25.0 7.6 43.3 33.7
tau243-254 300.5 653.0 705.9 276.9 838.1 48.5 230.5 94.5 393.5 297.6
tau260-267 76.2 180.7 192.7 74.3 255.3 14.6 55.9 30.1 110.0 87.7
tau275-281 41.7 100.9 105.0 28.3 121.7 7.8 30.1 14.5 56.3 45.4
tau281-290 78.3 188.8 204.4 80.1 254.7 13.5 59.3 25.5 113.1 90.2
MTBR tau282-290 171.0 543.2 480.6 139.1 493.5 37.5 53.7 73.5 249.0 217.7
tau tau299-317 251.6 1065.0 653.9 317.2 952.0 33.4 205.4 88.4 445.9 395.1
C[+57]tau322-340 704.4 2272.6 2061.6 702.5 3200.5 109.2 620.6 251.4 1240.4 1121.2
tau341-347 710.1 2772.5 1557.3 588.2 2096.4 28.1 570.4 230.3 1069.2 970.4
tau344-349 733.9 4551.6 1657.4 693.0 1634.8 115.0 826.2 242.9 1306.9 1426.7
tau354-369 877.3 2554.8 1652.7 923.9 2936.5 111.6 816.1 277.7 1268.8 1026.9
tau386-395 182.1 758.8 340.6 213.6 685.0 29.2 254.4 63.1 315.9 270.3
C-term tau396-406 19.2 92.3 29.5 28.0 48.0 1.9 15.2 4.2 29.8 29.3
tau tau407-438 1.3 3.1 3.2 1.2 4.3 1.7 0.8 0.2 2.0 1.4
tau407-441 39.8 37.9 57.4 28.2 91.8 0.0 5.2 0.0 32.5 31.8
Aβ28-42 231.8 826.2 779.9 774.1 787.7 3.9 110.2 39.5 444.2 377.9
Aβ28-40 20.0 13.3 3.8 157.5 55.5 0.2 74.7 6.1 41.4 54.0
Aβ16-27 250.3 760.9 704.4 1116.6 790.4 8.7 231.0 36.9 487.4 407.7
Aβ
Aβ1-15 10.3 21.4 21.9 39.6 38.4 0.4 10.3 1.1 17.9 15.2
Aβ4-15 12.0 19.2 23.9 29.2 27.4 0.4 9.4 2.3 15.5 11.1
Aβ3pGlu-15 6.1 13.6 11.1 27.9 18.2 0.2 7.2 0.7 10.6 9.3

4
Supplementary Figures
Figure legends

Figure S1. Schematic presentation of sarkosyl protocol.

Figure S2. (A) Scatter plot of all the proteins found in sarkosyl-insoluble pellet ranked in
decreasing order against their log2(iBAQ) protein abundance quantified using label-free
proteomics in AD brains following denaturing SDS-PAGE and in-gel digestion with trypsin.
Tau (MAPT) was one of the top proteins present, while iBAQ intensity indicated Aβ was not
as abundant after SDS-PAGE separation. (B) Bar plots for the MRM quantitative estimation
of MT-3, α-synuclein, β-synuclein, Aβ28-42, Aβ28-40 and tau243-254 peptides in AD starting
homogenate (SM), sarkosyl-soluble (SN) and -insoluble (P) fractions. The quantitative results
indicate enrichment of Aβ and tau peptides in the final sarkosyl-insoluble pellet.

Figure S3. Western blotting of (A) synthetic sAβ1-42 (2.2µg) and starting material (SM) and
sarkosyl soluble (SarkSol) fractions from AD and disease control (DCN) brains with Dako anti-
Tau (green) and anti-Aβ (WO2, red) antibodies; (B) DCN sarkosyl-insoluble fraction with
anti-TDP43 antibody.

Figure S4. Scatter plots of tau and Aβ peptides with mean ± SD in the (A) starting
homogenate, (B) sarkosyl-soluble, and (C) sarkosyl-insoluble fractions in AD (red) and
disease-control (DCN) (blue) brains. Scatter plots of mean ± SD concentrations of T181, T217
and their respective phosphorylated species pT181 and pT217 in the (D) starting material, (E)
sarkosyl-soluble, and (F) sarkosyl-insoluble fractions in AD (red) and disease-control (DCN)
(blue) brains.

Figure S5. Bar plots of log2-transformed quantification data from label-free quantitative
proteomics (normalized protein abundance iBAQ) with their respective error bars for all the
proteins depicted in the Figures 4B and C including Aβ, Tau (MAPT), Apolipoprotein E
(ApoE), Complement C4B, Complement C3, Small ribonucleoprotein E (SNRPE), clusterin
(CLU), U1 small ribonucleoprotein 70 kDa (SNRNP70), myosin light chain (MLY1), myosin
heavy chain (MYH1), Filamin-A (FLNA), α-synuclein (SNCA), and TDP43 (TARDBP) in
the AD (red) and disease control brains (DCN) (green) pellets, indicating significant
enrichment of these proteins in sarkosyl insoluble fraction of AD vs DCN. Neurofilament
light (NEFL) remained unchanged in the sarkosyl-insoluble pellet.

Figure S6. (A) Heatmap of the z-score normalized protein abundance (iBAQ) for all the
proteins found significantly (FDR < 0.05) enriched in sarkosyl-insoluble pellets of AD vs
DCN. (B) Heatmap of the z-score normalized protein abundance (iBAQ) for all the proteins
with FDR < 0.001 and significantly enriched in sarkosyl-insoluble pellets AD vs DCN.

Figure S7. Protein sequence coverage of ferritin (FRIH), tau (MAPT), TDP43 (TARDBP),
ApoE, α-synuclein (SNCA), SNRPE and neurofilament light (NEFL) from the label-free
proteomics quantification of the sarkosyl-insoluble pellets in AD and DCN brains. Only
methionine oxidation was used as variable modification. While tau had almost 90 %
sequence coverage, C-terminal intact peptides from TDP43 were not present in the sarkosyl
insoluble pellets.

Figure S8. Negative staining EM for 11 AD brain sarkosyl pellets (scale bar 50 nm), a
representative PHF filament from AD03 sample, and for the 1st and 2nd 16K g pellets from
AD11 sample showing scattered filamentous material found after an extended search.
5
Figure S9. Negative staining EM for sarkosyl insoluble fractions from disease-control (DCN)
ALS brains.

Figure S10. Negative immunogold staining EM for recombinant Aβ1-42 fibrils with (A)
WO2, after incubation with sarkosyl ~1% for 24h, (B) 6E10, and (C) 1E8 primary antibodies
and for recombinant hTau441 heparin-induced filaments with (D) TAU1, (E) HJ8.5 primary
antibodies. Secondary antibodies were 10nm-gold labelled anti-mouse antibodies.

Figure S11. Negative immunogold staining EM for AD04, AD06, AD09 and AD1 brain
samples with anti-tau primary monoclonal mouse (A-B) TAU1, (C) RNF5, (B) HJ8.5, and
polyclonal rabbit (E) Dako Tau K9JA and (F) p-tau (Ser202, Thr205) AT8 antibodies;
Secondary antibodies were 10nm-and 5nm-gold labelled anti-mouse and anti-rabbit
antibodies, respectively.

Figure S12. Negative immunogold staining EM for AD04, AD09, and AD11 brain samples
with primary anti-Aβ monoclonal mouse (A-B) WO2, (C-D) 6E10 antibodies incubated with
secondary anti-mouse 10nm-gold, and (E-F) monoclonal human anti-Aβ aducanumab
antibody incubated with secondary anti-human 6nm-gold labelled antibody.

Figure S13. Negative double immunogold staining EM for AD06 brain sample with (A)
primary monoclonal mouse tau RNF5 and human Aβ aducanumab antibodies and (B)
primary polyclonal tau K9JA and monoclonal Aβ 6F/3D antibodies. Secondary antibodies
were 10nm-gold anti-mouse for tau RNF5 (blue arrows in (A)) and for Aβ 6F/3D (orange
arrows in (B)), 6nm-gold anti-human for aducanumab (red arrows in (A)), and 5nm-gold anti-
rabbit for tau K9JA (cyan arrows in (B)). The red and orange arrows indicate Aβ, and the blue
and cyan arrows indicate tau aggregates in (A) and (B).

Figure S14. Cryo-EM imaging of (A) filaments and data analysis: (B) 2D class averages for
PHFs at ~7Å, (C) 3D refined map for PHFs at ~6Å, and (D) corresponding final (in red) FSC
(Fourier Shell Correlation) curve vs resolution (Å-1), (E) (B) tau PHF model (PDB:5osl) in
cyan with potentially ubiquitinated site K343 (R4) fitted to the cryo-EM map (pink net) of tau
filament at ~5.7 Å resolution (picture obtained using UCSF Chimera68), (F) (A) Negative
immunogold staining with polyclonal rabbit anti-ubiquitin antibody. Secondary antibody was
anti-rabbit 5nm-gold labelled antibodies. Scale bar 50 nm.

6
Figure S1.

7
Figure S2.

SM SN P

7
 20
25

20
25
2

2
10
15
37
50
75

10
15
37
50
75
5

5
100
150
250

100
150
250
CN09-SM A

B
CN01-SM
CN09-SarkSol
CN01-SarkSol
Figure S3.

CN05-SM CN06-SM

CN05-SarkSol CN06-SarkSol

CN02-SM CN03-SM

CN03-SarkSol
CN02-SarkSol

AD04-SM AD03-SM

AD04-SarkSol AD03-SarkSol

AD08-SM
AD09-SM
AD08-SarkSol
AD09-SarkSol
sAβ1-42
sAβ1-42

20
25

2
10
15
50
20
25

37
75

5
2

100
150
250
37
50
75

10
15

5
100
150
250

DCN10-SM AD07-SM

DCN10-SarkSol AD07-SarkSol

AD10-SM
DCN04-SM
AD10-SarkSol
DCN04-SarkSol
DCN07-SM
AD02-SM
DCN07-SarkSol
AD02-SarkSol
DCN08-SM
AD06-SM
DCN08-SarkSol
AD06-SarkSol

AD05-SM
AD01-SM
AD05-SarkSol
AD01-SarkSol
sAβ1-42
sAβ1-42

8
Figure S4.

9
Figure S5.

10
Figure S6.

11
Figure S7.

12
Figure S8.

AD01 AD02 AD03

AD04 AD05 AD06

AD07 AD08 AD09

AD10 AD11 PH
F

AD11, 1st 16K g AD11, 2nd 16K g

13
Figure S9.

DCN01 DCN02 DCN03

DCN04 DCN05 DCN06

DCN07 DCN08 DCN09

DCN10

14
Figure S10.
F
A Aβ42 WO2 B Aβ42 6E10

C Aβ42 1E8 D hTau441 TAU1

E hTau441 HJ8.5

15
Figure S11.

A AD11 TAU1 B AD09 TAU1

C AD04 RNF5 D AD11 HJ8.5

E AD06 K9JA F AD09 AT8

16
Figure S12.

A AD04 WO2 B AD09 WO2

C D

C AD04 6E10 D AD11 6E10

E AD09 Adu F AD09 Adu

17
Figure S13.

A RNF5 (blue)+Adu (red)

B K9JA (blue)+6F/3D (orange)

18
Figure S14.

A B

C D

Ubq
E F
K343

19