1

SUPPORTING INFORMATION

Site-specific characterization of threonine, serine and

tyrosine glycosylations of amyloid precursor

protein/amyloid β-peptides in human cerebrospinal fluid

Adnan Halim, Gunnar Brinkmalm, Ulla Rüetschi, Ann Westman-Brinkmalm, Erik

Portelius, Henrik Zetterberg, Kaj Blennow, Göran Larson* and Jonas Nilsson*

goran.larson@clinchem.gu.se

jonas.nilsson@clinchem.gu.se

Content

Fig. S1-S10

Table S1 and S2

  2

VNGEFSLDDLQPWHSFGADSVPANTENEVEPVDARPAADRGLTTRPGSGLTNIKTEEISEVKMDAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA

–25

–51–57

–63

–10

APP/A! X-15

1 421516

1817

1920

–3 15154174A!1-X

" #15

–20–30–40–50 20 30–60 YYYYYYYYY

TTT

TT T

TT

T S

15

11111

15TT T T STT T T S

15T T S

!

175 Y

–45 T 15TT T T S

1 281 301 331 341 371 381 391 401 42

Fig. S1. Amyloid Precursor Protein (APP) amino acid sequence containing the

Aβ1-X and APP/AβX-15 glycopeptides identified in human CSF. The positions of

the α−, β− and γ-secretase cleavage sites are shown as arrowheads above the APP

amino acid sequence. The APP/AβX-15 glycopeptides, all ending at Asn15, are

shown above the sequence (blue series). The Aβ1-X glycopeptides are shown below

the sequence (green series). The glycosylation sites presented in this study are

indicated as red S/T/Y residues. Longer Aβ1-X peptides could not be detected in

glycosylated form (black series).

  3

15 20 25 30 35 40Time (min)

500 600 700 800

457.454+/459.954+

609.603+

566.923+

675.804+

612.943+

694.294+

767.064+

700 800 900 1000 1100 1200m/z

866.455+

722.216+

1082.824+

877.045+A!1-17

A!1-15

A!1-15/A!1-15*

A!1-15

SA2-A!1-1516.7-18.2 min

SA2-A!4-17

Inte

nsity

A!1-15*

33.8-35.3 min

A!1-40

A!1-40

A!1-40

A!1-4015.0-40.0 min

A!1-39

A!1-38

A!1-34

A!1-14

APP/A!"25-15

m/z

A B

C

14 16 18 20 22 24 26Time (min)

Intensity

A!1-15A!1-15*

SA2-A!1-15SA3-A!1-15

D

b

b

b

SA3-A!1-15

a a a a a

877.045+

Intensity

Fig. S2. Immunopurified Aβ peptides and glycopeptides in human CSF. (A)

Total-ion current chromatogram at 15.0-40.0 min of immunopurified Aβ peptides and

glycopeptides. (B) Combined full mass spectra at 33.8-35.3 min showing Aβ1-40 and

an apparent lack of Aβ1-40 glycopeptides. (C) Combined full mass spectra at 16.7-

18.2 min showing Aβ1-15, SA2-Aβ1-15 and SA3-Aβ1-15, as well as additional Aβ

peptides and glycopeptides. Minor ions(a) and singly charged ions(b) are of unknown

origin. (D) Extracted ion chromatograms for the m/z values corresponding to [M +

4H]4+ of the Aβ1-15 peptide, Aβ1-15-Arg-13C15N standard (Aβ1-15*), SA2-Aβ1-15,

and SA3-Aβ1-15. Parent ions are in the [M + zH]z+ form and charges are shown in

superscript when z >1.

  4

200 400 600 800 1000

542.94+

723.63+

626.53+

572.73+

470.14+

669.53+

274.0757.02+ 858.02+

504.8292.0 939.12+429.74+

Inte

nsity

A!4-15 MS2 615.514+

200 400 600 800 1000

723.53+

626.83+

572.63+

814.63+

547.0

428.9

669.73+939.42+340.9 756.62+

529.0

858.12+274.0 355.0

565.0

A!4-15 MS2 683.784+

611.34+

-H2O

B

A A!1-15 MS2 621.514+

200 400 600 800 1000 1200

548.94+

508.44+ 575.9731.43+

274.0

Inte

nsity

621 622 623

621.76

622.01621.51

622.26

Z=4

677.53+

Ac

Neu5Ac, 291 Da

O-AcNeu5Ac, 333 Da

Lactonized Neu5AcNeu5Ac, 565 Da

Hex 162 DaHexNAc 203 DaA! peptide

A! 1-16 MS2 799.084+

653.84+

774.03+

400 600 800 1000 1200

871.13+

726.34+

968.03+

291.9 581.24+

400 800 1000 1200

774.13+

871.23+

722.04+

565.1529.2

600

A! 1-16 MS2 794.584+

C

D E

Inte

nsity

m/z m/z

G

400 800 1200 1600 2000

609.44+

811.73+568.84+

757.83+

A!1-17 MS2 681.804+

F

400 600 800 1000 1200

711.44+

638.54+

851.13+729.53+

597.94+

947.93+797.13+

274.0 1093.62+1195.02+

A! -3-15 MS2 783.844+

894.13+671.34+

Inte

nsity

-H2O

-H2O

-2H2O

-H2O

  5

400 800 1200 1600 2000

811.703+

681.804+

609.034+

908.743+

1034.482+

1136.022+

Inte

nsity

812.04811.70

812.37

812.71

813.04

757.693+

A!1-17 MS2 754.584+

855.053+

400 600 800 1000 1200

909.23+

754.84+

811.73+

682.04+

609.34+1034.92+274.0

1217.02+

292.0

Inte

nsity

A! 1-17 MS2 827.354+

400 600 800 1000 1200

909.13+

812.03+

754.94+

682.04+

609.14+316.01006.03+

1217.82+

274.3

334.0

A! 1-17 MS2 837.864+

Ac

Ac

400 600 800 1000 1200

811.93+

909.33+758.33+

750.44+

547.0

1035.22+

529.1 1217.42+1136.32+690.43+

565.0

1000.1

A! 1-17 MS2 822.854+

H I

J

200 400 600 800 1000 1200

603.34+

530.34+ 707.03+

804.23+

653.03+

490.04+

274.0 328.8

A! 4-17 MS2 675.804+

291.9

L

200 400 600 800 1000 1200

m/z

803.83+

676.04+

603.24+901.03+

292.01059.42+531.04+

707.13+

A! 4-17 MS2 748.584+

836.5

Inte

nsity

M

K

m/z

O

200 400 600 800 1000 1200

634.14+706.84+

845.13+

761.7593.64+ 942.33+274.2

A! 1-18 MS2 779.364+

Inte

nsity

N

A! 5-17 MS2 639.034+

200 400 600 800 1000 1200

566.54+

754.73+

658.23+

493.74+

453.54+274.0

700.73+

603.83+

-H2O-H2O

-2H2O

-H2O-H2O

-H2O

-H2O

-H2O

  6

400 600 800 1000 1200 1400

816.44+

991.13+

743.74+

671.14+840.23+

1157.62+

274.0 1088.33+ 1259.72+920.3 1340.62+

565.1

583.2547.2

772.53+

A! 1-19 MS2 888.894+

400 600 800 1000 1200 1400

840.13+

991.23+

743.64+

670.84+

1158.22+

879.5316.1 1259.42+1340.62+

816.44+

334.2

A! 1-19 MS2 899.394+

Ac

Ac

400 600 800 1000 1200 1400

670.94+

743.64+

1282.6

342.31410.7

894.13+630.64+ 1264.5

A! 1-19 MS2 816.124+

Inte

nsity

Inte

nsity

P Q

400 600 800 1000 1200

942.33+779.64+

791.13+

768.4

706.94+

633.84+

1084.72+

567.3274.1 1186.22+

1039.13+

485.3 1267.52+367.2

650.6

A! 1-18 MS2 852.134+

Ac

400 600 800 1000 1200

844.83+942.33+

779.54+

706.94+

634.24+316.21084.62+

1267.72+

A! 1-18 MS2 862.634+

274.2

334.0

791.33+

Ac

400 600 800 1000 1200

775.34+

790.83+

942.33+

1084.22+547.1

1185.82+918.5723.33+529.0 1033.2

1267.32+

565.0

A! 1-18 MS2 847.624+

Inte

nsity

R S

T U

400 600 800 1000 1200m/z

707.84+

780.54+

812.3 953.5

1082.4667.34+

533.3 632.4

1038.83+

972.8

920.6889.33+

A! 1-20 MS2 852.894+

Inte

nsity

565.0

400 600 800 1000 1200 1400m/z

894.23+

990.93+812.34+

670.9 1158.2 1282.4529.1

1410.6

547.1

840.33+

A! 1-19 MS2 884.384+

V W

888.33+

-H2O

-H2O -H2O

-H2O

-2H2O

-H2O

-H2O-2H2O -H2O

-H2O-2H2O

Fig. S3. CID MS2 spectra of Aβ1-X glycopeptides. (A) MS1 (left) and CID MS2

(right) of SA-Aβ1-15. (B) CID MS2 of SA2-Aβ4-15. (C) CID MS2 of lactonized SA3-

Aβ4-15. The presence of the peptide+Neu5AcHexNAc fragment ion at m/z 669.7

demonstrates that the disialosyl group was attached to the Hex rather than to the

  7

HexNAc. (D) CID MS2 spectrum of SA3-Aβ1-16. (E) CID MS2 spectrum of

lactonized SA3-Aβ 1-16. (F) CID MS2 spectrum of SA2-Aβ –3-15. (G) MS2 spectrum

of SA-Aβ1-17 (H) MS2 spectrum of SA2-Aβ1-17. This MS2 spectrum was acquired in

the ion cyclotron resonance (ICR) mode. The isotopic resolution of the fragment ions

is demonstrated in the insert. The sensitivity in ICR mode is lower than for the linear

quadrupole ion trap (LQIT) mode, which was used for the collection of most CID

MS2 spectra. (I) - (W) Additional CID MS2 spectra of Aβ1-X glycopeptides. Ions are

in the [M + zH]z+ form and the charges are shown in superscript when z>1. The mass

accuracy was <5 ppm for all precursor ions and their monoisotopic m/z values and

charge (4+) are shown. The annotated m/z values of fragment ions are those picked by

the Xcalibur program. The Neu5Ac oxonium ion and its loss of H2O are present at

m/z 292 and 274, respectively. The O-AcNeu5Ac oxonium ion and its loss of H2O are

present at m/z 334 and 316, respectively. The lactonized Neu5AcNeu5Ac oxonium

ion and its loss of one and two H2O are present at m/z 565, 547 and 529, respectively.

  8

DAEFRHDSGY(947.3230)EVHHQc c+2 z z+2

--- --- 1 D 15 --- ---204.0979 102.5526 2 A 14 2643.0614 1322.0344333.1405 167.0739 3 E 13 2572.0243 1286.5158480.2089 240.6081 4 F 12 2442.9817 1221.9945636.3100 318.6586 5 R 11 2295.9133 1148.4603773.3689 387.1881 6 H 10 2139.8122 1070.4097888.3959 444.7016 7 D 9 2002.7533 1001.8803975.4279 488.2176 8 S 8 1887.7263 944.36681032.4493 516.7283 9 G 7 1800.6943 900.85082142.8357 1071.9215 10 Y(947.3230) 6 1743.6729 872.34012271.8783 1136.4428 11 E 5 633.2865 317.14692370.9467 1185.9770 12 V 4 504.2439 252.62562508.0056 1254.5064 13 H 3 405.1755 203.09142645.0645 1323.0359 14 H 2 268.1166 134.5619--- --- 15 Q 1 131.0577 66.0325

A

DAEFRHDSGY(947.3230)EVHHQKLc c+2 z z+2

--- --- 1 D 17 --- ---204.0979 102.5526 2 A 16 2884.2405 1442.6239333.1405 167.0739 3 E 15 2813.2033 1407.1053480.2089 240.6081 4 F 14 2684.1608 1342.5840636.3100 318.6586 5 R 13 2537.0923 1269.0498773.3689 387.1881 6 H 12 2380.9912 1190.9993888.3959 444.7016 7 D 11 2243.9323 1122.4698975.4279 488.2176 8 S 10 2128.9054 1064.95631032.4493 516.7283 9 G 9 2041.8733 1021.44032142.8357 1071.9215 10 Y(947.3230) 8 1984.8519 992.92962271.8783 1136.4428 11 E 7 874.4656 437.73642370.9467 1185.9770 12 V 6 745.4230 373.21512508.0056 1254.5064 13 H 5 646.3545 323.68092645.0645 1323.0359 14 H 4 509.2956 255.15152773.1231 1387.0652 15 Q 3 372.2367 186.62202901.2180 1451.1127 16 K 2 244.1781 122.5927--- --- 17 L 1 116.0832 58.5452

B

DAEFRHDSGY(365.1322)EVHHQc c+2 z z+2

133.0608 67.0340 1 D 15 --- ---204.0979 102.5526 2 A 14 2060.8706 1030.9390333.1405 167.0739 3 E 13 1989.8335 995.4204480.2089 240.6081 4 F 12 1860.7909 930.8991636.3100 318.6586 5 R 11 1713.7225 857.3649773.3689 387.1881 6 H 10 1557.6214 779.3143888.3959 444.7016 7 D 9 1420.5625 710.7849975.4279 488.2176 8 S 8 1305.5355 653.27141032.4493 516.7283 9 G 7 1218.5035 609.75541560.6449 780.8261 10 Y(365.1322) 6 1161.4821 581.24471689.6875 845.3474 11 E 5 633.2865 317.14691788.7559 894.8816 12 V 4 504.2439 252.62561925.8148 963.4110 13 H 3 405.1755 203.09142062.8737 1031.9405 14 H 2 268.1166 134.5619--- --- 15 Q 1 131.0577 66.0325

925.383+

828.683+

636.31

333.14

1254.522+

c3

694.284+

268.12z2

504.24z4

c5

M

M-Neu5Ac773.37c6 c13

1136.452+c11

1186.492+c12+1

480.21

z3

c4

z5

Intensity

200 400 600 800 1000 1200

M

911.703+

882.363+

1032.45

872.352+

975.43

M-Ac

c9 1071.922+c10

z6

c81001.892+

z9c14

888.40c7 M-2Neu5Ac

1096.472+

-H2O

850 900 950 1000 1050 1100

Intensity

D A E F RHDSGYEVHHQc3 c4 c5 c6 c7 c8 c9 c10c11c12

z2z3z4

c13c14

A!1-15 z9 z5z6

405.18

633.29

M

SA2

DAE FRHDSGYEVHHQK Lc3 c4 c5 c6 c7 c8 c9 c10c11c12

z2z3z4z7

c13 c14c15

z8z9A!1-17 z11 z5z6

SA2

908.733+

1071.922+636.31

333.14c3

c5c10

c4

z2z3 z4

244.18372.24

480.21

509.30

M-NeuAc

M-H2O1000.103+

z5

c6

z7

c7

z6

200 400 600 800 1000 1200 1400

m/z

745.42

M 754.584+ 1005.773+M

874.47

980 990 1000 1010 1020 1030

1032.45975.43 986.103+

976.43

1021.44z=2

1033.45

992.10992.43992.77993.10

991.763+M-Ac

c8c9

M991.76

1021.941022.44

z9M-Ac-H2O

z=3

z=2992.93*

993.43*993.94*

z8

c15

M-2NeuAc-H2O

z11c11c12

z10

1209.552+

1254.512+c13

c14

z10

200 400 600 800 1000m/z

688.303+780.832+

581.242+

1032.44

963.412+

480.21888.40

268.12333.14

Intensity

z2c3

c4

z6

c5z5

c14

z8

548.734+ 731.643+M M

773.37c6

c9

c13c7

c12

c10

M-AcM-Ac-H2O

c11z11

D A E FRHDSGYEVHHQc3 c4 c5 c6 c7 c9 c10c11c12

z2z3z4

c13c14

A!1-15z11 z5z6

HexHexNAc

z4

C

m/z

9

D DAEFRHDSGY(365.1322)EVHHQKL

c c+2 z z+2

133.0608 67.0340 1 D 17 --- ---204.0979 102.5526 2 A 16 2302.0497 1151.5285333.1405 167.0739 3 E 15 2231.0125 1116.0099480.2089 240.6081 4 F 14 2101.9700 1051.4886636.3100 318.6586 5 R 13 1954.9015 977.9544773.3689 387.1881 6 H 12 1798.8004 899.9039888.3959 444.7016 7 D 11 1661.7415 831.3744975.4279 488.2176 8 S 10 1546.7146 773.86091032.4493 516.7283 9 G 9 1459.6825 730.34491560.6449 780.8261 10 Y(365.1322) 8 1402.6611 701.83421689.6875 845.3474 11 E 7 874.4656 437.73641788.7559 894.8816 12 V 6 745.4230 373.21511925.8148 963.4110 13 H 5 646.3545 323.68092062.8737 1031.9405 14 H 4 509.2956 255.15152190.9323 1095.9698 15 Q 3 372.2367 186.62202319.0272 1160.0173 16 K 2 244.1781 122.5927--- --- 17 L 1 116.0832 58.5452

200 400 600 800 1000 1200 1400

Intensity

1016.743+

773.37

925.383+

1242.012+1096.472+

333.14

268.12

480.21636.31

888.40z2

c3c4 c5 c6

c7

M-NeuAc 1022.413+M

M-H2O

M-Ac1008.403+

1017.07

1017.41

1018.07

1016.74 1017.74 1017.88z6

767.064+

c91032.45 1217.472+

c10

1331.542+c12

405.18z3 504.24

z4

633.29z5 M-2NeuAc

M-3NeuAc

1147.432+z9

M

DAE FRHDSGYEVHHQc3 c4c5 c6 c7 c9 c10 c12

z2z3z4z6A!1-15

z5z9

M-H2OSA3

1018.38z6

DAEFRHDSGY(1238.4184)EVHHQc c+2 z z+2

--- --- 1 D 15 --- ---204.0979 102.5526 2 A 14 2934.1568 1467.5821333.1405 167.0739 3 E 13 2863.1197 1432.0635480.2089 240.6081 4 F 12 2734.0771 1367.5422636.3100 318.6586 5 R 11 2587.0087 1294.0080773.3689 387.1881 6 H 10 2430.9076 1215.9574888.3959 444.7016 7 D 9 2293.8487 1147.4280975.4279 488.2176 8 S 8 2178.8217 1089.91451032.4493 516.7283 9 G 7 2091.7897 1046.39852433.9311 1217.4692 10 Y(1238.4184) 6 2034.7683 1017.88782562.9737 1281.9905 11 E 5 633.2865 317.14692662.0421 1331.5247 12 V 4 504.2439 252.62562799.1010 1400.0541 13 H 3 405.1755 203.09142936.1599 1468.5836 14 H 2 268.1166 134.5619--- --- 15 Q 1 131.0577 66.0325

E

300 500 700 900 1100

1032.44

874.46

1095.97

636.31

333.14 480.21244.18 509.29

c3 c4z2 z4

z3372.24

609.034+ 812.033+M M

c5

z5

700 740 780 820

773.37

701.832+

831.382+845.352+730.352+

744.343+

768.693+

M780.832+c10

c6

773.683+c16

773.862+z10

z8

v

z9

z15

z16

646.35

M-H2O

M-Ac-H2O

M-Ac

z11 c11

z6

z7

888.39c7

c9

c13

c8975.42

z13

c12

z14

c15

c14z12

m/z

D A E F R H D S G Y E V H H Q K Lc3 c4 c5 c6 c7 c8 c9

c10c11c12

z2z3z4z7

c13 c14c15

z8z9

A!1-17z11 z5z6z10

HexHexNAc

c16

z12z13z14z16z15

200 400 600 800 1000 1200 1400 1600 1800m/z

1102.803+

1005.773+

874.47

1217.472+

773.37

636.31509.30

1364.12372.24244.18

745.43

1625.22

D A E F R H D S G Y E V H H Q K Lc3 c4 c5 c6 c7 c8 c9 c10

z2z3z4z7

c13

z8A! 1-17

c10

Inte

nsity

z2 z3

z4

z6

z6

z7

1138.482+z8

1268.53333.14c3 480.21

c4

c5

c6

M

M

888.40c7

975.43c8

1400.062+

c13

1532.622+

827.354+

c15

M-NeuAc

c9

c15

F

c16

980 1000 1020 1040 1060 1080 1100 1120 1140

1088.483+

1005.773+

1032.45975.43

1058.763+

1000.441036.03 1138.482+

1109.06 z8c8

c9

Inte

nsity

1102.803+M

M-AcM-H2O

1097.143+

M-NeuAc

1065.123+c+116

c+116-H2O

m/z

DAEFRHDSGY(1238.4184)EVHHQKLc c+2 z z+2

--- --- 1 D 17 --- ---204.0979 102.5526 2 A 16 3175.3359 1588.1716333.1405 167.0739 3 E 15 3104.2987 1552.6530480.2089 240.6081 4 F 14 2975.2562 1488.1317636.3100 318.6586 5 R 13 2828.1877 1414.5975773.3689 387.1881 6 H 12 2672.0866 1336.5470888.3959 444.7016 7 D 11 2535.0277 1268.0175975.4279 488.2176 8 S 10 2420.0008 1210.50401032.4493 516.7283 9 G 9 2332.9687 1166.98802433.9311 1217.4692 10 Y(1238.4184) 8 2275.9473 1138.47732562.9737 1281.9905 11 E 7 874.4656 437.73642662.0421 1331.5247 12 V 6 745.4230 373.21512799.1010 1400.0541 13 H 5 646.3545 323.68092936.1599 1468.5836 14 H 4 509.2956 255.15153064.2185 1532.6129 15 Q 3 372.2367 186.62203192.3134 1596.6604 16 K 2 244.1781 122.5927--- --- 17 L 1 116.0832 58.5452

SA3m/z

10

F R H D S G Y E V H H Q K Lc2 c3 c4 c5 c6 c7 c8 c9

c10 c11c12

z2z3z4z7

219.13 321.20c2 372.24

z3

509.30z4

458.26

573.29c4

c3

244.18z2

660.32

675.804+

717.35

c5

803.703+

887.073+M-Ac

874.47z7

914.372+c7

901.403+

200 400 600 800 1000 1200

M

M

978.902+c8

c9

1028.422+

1059.512+

1165.492+

c11

1096.962+c10

1229.522+

c12

c6

M-NeuAcM-2NeuAcM-2NeuAc-H2O

H

Inte

nsity

A! 4-17 FRHDSGY(947.3230)EVHHQKLc c+2 z z+2

--- --- 1 F 14 --- ---321.2034 161.1053 2 R 13 2537.0923 1269.0498458.2623 229.6348 3 H 12 2380.9912 1190.9993573.2892 287.1482 4 D 11 2243.9323 1122.4698660.3212 330.6643 5 S 10 2128.9054 1064.9563717.3427 359.1750 6 G 9 2041.8733 1021.44031827.7290 914.3682 7 Y(947.3230) 8 1984.8519 992.92961956.7716 978.8894 8 E 7 874.4656 437.73642055.8400 1028.4237 9 V 6 745.4230 373.21512192.8989 1096.9531 10 H 5 646.3545 323.68092329.9579 1165.4826 11 H 4 509.2956 255.15152458.0164 1229.5119 12 Q 3 372.2367 186.62202586.1114 1293.5593 13 K 2 244.1781 122.5927--- --- 14 L 1 116.0832 58.5452

SA2

DAEFRHDSGYEVHHQc c+2 z z+2

--- --- 1 D 15 --- ---204.0979 102.5526 2 A 14 1695.7384 848.3729333.1405 167.0739 3 E 13 1624.7013 812.8543480.2089 240.6081 4 F 12 1495.6587 748.3330636.3100 318.6586 5 R 11 1348.5903 674.7988773.3689 387.1881 6 H 10 1192.4892 596.7482888.3959 444.7016 7 D 9 1055.4303 528.2188975.4279 488.2176 8 S 8 940.4033 470.70531032.4493 516.7283 9 G 7 853.3713 427.18931195.5127 598.2600 10 Y 6 796.3499 398.67861324.5553 662.7813 11 E 5 633.2865 317.14691423.6237 712.3155 12 V 4 504.2439 252.62561560.6826 780.8449 13 H 3 405.1755 203.09141697.7415 849.3744 14 H 2 268.1166 134.5619--- --- 15 Q 1 131.0577 66.0325

200 400 600 800 1000 1200 1400 1600m/z

848.382+

773.37

636.31

268.12

748.332+

1560.691055.431195.52

940.41504.25

405.18 1423.63

D A E F R H D S G Y E V H H Qc4 c5 c6 c7 c9 c10 c12

z2z3z4z7

A! 1-15z5z9

z2

z3

z4c5

480.21c4

z14

c6

z12

z8

z9 c10 c12

c13

1324.56c11

1348.59z11975.43

c8

c8 c11 c13c14

z8z10z11z12z13z14

609.603+M

913.902+M

Inte

nsity

G

c13

862.713+

c13

z5

z5

z6

z6

980 990 1000 1010 1020 1030m/z

978.902+

1021.452+

1028.422+

986.54 993.442+

997.35

c9

c8

z9

z8+1

z9 z8

z10

z10z11

z11

A! -3-15

V K M D A E F R H D S G Y E V H H Qc3 c4 c6 c7 c9 c10 c11 c13 c14 c16c15

z2z3z5I

268.12

219.14

491.26

562.30

633.29

691.35405.18

783.834+ 1044.783+

838.41

948.083+1001.763+M-NeuAc

MM

1044.783+M-Ac

1131.57

1246.60

1251.032+

1333.63

1365.082+

1433.612+

z2

z3 z5

c3376.24

c4

c6

c7

c17

c9

c10

c11

c13

c15

c16

200 400 600 800 1000 1200 1400 1600 1800m/z

Inte

nsity

VKMDAEFRHDSGY(947.3230)EVHHQc c+2 z z+2

--- --- 1 V 18 --- ---245.1972 123.1022 2 K 17 3017.2238 1509.1155376.2377 188.6225 3 M 16 2889.1289 1445.0681491.2646 246.1360 4 D 15 2758.0884 1379.5478562.3017 281.6545 5 A 14 2643.0614 1322.0344691.3443 346.1758 6 E 13 2572.0243 1286.5158838.4128 419.7100 7 F 12 2442.9817 1221.9945994.5139 497.7606 8 R 11 2295.9133 1148.46031131.5728 566.2900 9 H 10 2139.8122 1070.40971246.5997 623.8035 10 D 9 2002.7533 1001.88031333.6317 667.3195 11 S 8 1887.7263 944.36681390.6532 695.8302 12 G 7 1800.6943 900.85082501.0395 1251.0234 13 Y(947.3230) 6 1743.6729 872.34012630.0821 1315.5447 14 E 5 633.2865 317.14692729.1505 1365.0789 15 V 4 504.2439 252.62562866.2095 1433.6084 16 H 3 405.1755 203.09143003.2684 1502.1378 17 H 2 268.1166 134.5619--- --- 18 Q 1 131.0577 66.0325

SA2

11

K

204.10

333.14

362.22480.21

490.31560.802+

618.37

636.31

651.973+

662.782+

670.842+

689.663+

780.842+

840.902+

849.372+

888.39 892.49

913.402+

c2

c3

z3

z4

c4

z5

c5

c16

c11

z11

c17

722.683+

c18

c13

z14

c14

c7 z7

c15

975.42c8

c9c10

1195.51

c12 c16z16

c18

579.284+ 772.033+

z6

z9

z17

D A E F R H D S G Y E V H H Q K LV Fc3 c4 c5 c7 c8 c9c10

z4z5z6z9

c13

A! 1-19

c15c16c2 c11c12 c14 c18

z3z7z11z14z16z17

M M

200 400 600 800 1000 1200m/z

Inte

nsity

1032.45

DAEFRHDSGYEVHHQKLVFc c+2 z z+2

--- --- 1 D 19 --- ---204.0979 102.5526 2 A 18 2183.0543 1092.0308333.1405 167.0739 3 E 17 2112.0172 1056.5122480.2089 240.6081 4 F 16 1982.9746 991.9909636.3100 318.6586 5 R 15 1835.9062 918.4567773.3689 387.1881 6 H 14 1679.8051 840.4062888.3959 444.7016 7 D 13 1542.7461 771.8767975.4279 488.2176 8 S 12 1427.7192 714.36321032.4493 516.7283 9 G 11 1340.6872 670.84721195.5127 598.2600 10 Y 10 1283.6657 642.33651324.5553 662.7813 11 E 9 1120.6024 560.80481423.6237 712.3155 12 V 8 991.5598 496.28351560.6826 780.8449 13 H 7 892.4914 446.74931697.7415 849.3744 14 H 6 755.4325 378.21991825.8001 913.4037 15 Q 5 618.3735 309.69041953.8950 977.4512 16 K 4 490.3150 245.66112066.9791 1033.9932 17 L 3 362.2200 181.61362166.0475 1083.5274 18 V 2 249.1359 125.0716--- --- 19 F 1 150.0675 75.5374

Inte

nsity

333.14219.14

480.21

490.32

618.37

636.31

773.37755.43

1032.45

z4

z5

z6

c3

c4

c5

c6

c9

888.894+M

1185.183+M

1170.853+M-Ac

1087.823+M-NeuAc

1135.503+

c18

1400.052+c13

D A E F R H D S G Y E V H H Q K LV Fc4c5 c6 c9

z6A! 1-19

c13c3

z4z5

c18

200 400 600 800 1000 1200 1400 1600 1800m/z

1485.662+1632.232+

M-2NeuAc M-NeuAc

1623.722+M-NeuAc-17

J DAEFRHDSGY(1238.4184)EVHHQKLVF

c c+2 z z+2

--- --- 1 D 19 --- ---204.0979 102.5526 2 A 18 3421.4727 1711.2400333.1405 167.0739 3 E 17 3350.4356 1675.7214480.2089 240.6081 4 F 16 3221.3930 1611.2001636.3100 318.6586 5 R 15 3074.3246 1537.6659773.3689 387.1881 6 H 14 2918.2235 1459.6154888.3959 444.7016 7 D 13 2781.1645 1391.0859975.4279 488.2176 8 S 12 2666.1376 1333.57241032.4493 516.7283 9 G 11 2579.1056 1290.05642433.9311 1217.4692 10 Y(1238.4184) 10 2522.0841 1261.54572562.9737 1281.9905 11 E 9 1120.6024 560.80482662.0421 1331.5247 12 V 8 991.5598 496.28352799.1010 1400.0541 13 H 7 892.4914 446.74932936.1599 1468.5836 14 H 6 755.4325 378.21993064.2185 1532.6129 15 Q 5 618.3735 309.69043192.3134 1596.6604 16 K 4 490.3150 245.66113305.3975 1653.2024 17 L 3 362.2200 181.61363404.4659 1702.7366 18 V 2 249.1359 125.0716--- --- 19 F 1 150.0675 75.5374

SA3

Fig. S4. ECD MS2 spectra of Aβ1-X glycopeptides (A)-(D) Fully m/z annotated ECD

spectra and lists of c- and z-ions accompanying Fig. 2 of the main text. (E) ECD MS2

spectrum of SA3-Aβ1-15. The presence of a glycosylated z6 peak is shown in the insert. (F)

ECD MS2 spectrum of SA3-Aβ1-17. The presence of a glycosylated z8 peak is shown in the

expansion. (G) ECD MS2 spectrum of Aβ1-15 peptide. (H) ECD MS2 spectrum of SA2-Aβ4-

17. The presence of glycosylated z8 and z9 peaks is shown in the expansion (I) ECD MS2

spectrum of SA2-Aβ–3-15. (J) ECD MS2 spectrum of SA3-Aβ1-19. (K) ECD MS2 spectrum

of Aβ1-19 peptide. The lists of c- and z-series of peptide backbone fragments were prepared

by the use of the MS product utility at the Protein Prospector homepage

(http://prospector.ucsf.edu/prospector/cgi-bin/msform.cgi?form=msproduct). The identified

fragment ions are annotated in the spectra, and shown in relation to the peptide sequences.

The molecular ion and its charged reduced forms (M) are present in the spectra. M-Ac, loss of

acetyl from M. The peaks were expanded and the M peaks were cropped.

12

400 800 1200 1600 2000

1038.37+

996.87+

1112.66+

953.81161.06+

1209.26+

–45-15 MS2 1079.367+

y57

y57y57

A

m/z

1079 1080 1081

1079.94

1079.79

1080.22

Z=7

1079.65

1080.37

1079.50

1079.36

Neu5Ac, 291 Da

Hex, 162 Da

HexNAc, 203 Da

peptidey57 peptide fragment

Inte

nsity

400 800 1200 1600 2000

1240.77+

1199.07+

1282.37+

–51-15 MS2 1323.177+

1323 1324 1325

1323.89

1323.741324.03

1324.171323.60

1323.461324.32

1324.461323.31

1324.60

1323.17

Inte

nsity

Z=7

400 800 1200 1600 2000

1094.77+

1136.47+

1018.21222.9

–51-15 MS2 1177.267+

Inte

nsity

C D

500 1000 1500 2000

1094.77+

1223.16+

–51-15 MS2 1135.677+

800 1000 1200 1400 1600 1800

1122.38+

1158.88+1085.68+

1049.28+

1282.96+

1590.19

1218.86+

–51-15 MS2 1194.418+

987.4

1331.66+

E

b9

500 1000 1500 2000m/z

1147.07+

1188.67+

1123.87+

1277.16+

–51-15 MS2 1229.427+

F

Inte

nsity

B

m/z

13

H

400 800 1200 1600 2000

1334.67+

1292.97+

1376.17+

1251.57+

–51-15 MS2 1417.067+ (*)

400 800 1200 1600 2000

1192.17+

1335.05+1112.46+

–57-15 MS2 1233.017+In

tens

ity

y57 y57

G

I

400 800 1200 1600 2000

1192.37+

1233.87+

1334.85+

1392.85+

–57-15 MS2 1274.757+ (*)

y57 y57

Inte

nsity

400 800 1200 1600 2000

1253.18+

1289.58+

1343.66+1216.68+834.892+

1392.36+

1441.06+

1668.8

1489.36+

–57-15 MS2 1325.228+ (*)

b15

b15

1180.28+

y57

y57

Inte

nsity

J

–63-15 MS2 1511.137+ (*)

600 1000 1400 1800

1428.77+

1470.47+1387.17+

1481.2

K

Inte

nsity

400 800 1200 1600 2000

1296.08+

1332.38+

1368.78+

1259.68+

1152.32+

b21

–63-15 MS2 1404.398+

L

m/zm/z

Fig. S5. CID MS2 of additional APP/AβX-15 glycopeptides. (A) MS1 and CID MS2 of

Neu5AcHex(Neu5Ac)HexNAc APP/Aβ–45-15 showing the diagnostic y57 peptide fragment

peaks. (B) MS1 and CID MS2 of (Neu5AcHexHexNAc)3 APP/Aβ–51-15. The same

glycopeptide possessing different charges, are fragmented here (z = 7) and in Fig. 4C of the

main text (z = 8). The diagnostic b9 and y57 ion peaks were only visible for z = 8, and were

generally more pronounced at the higher charge state of the APP/AβX-15 glycopeptides. (C) -

(L) CID MS2 spectra of additional APP/AβX-15 glycopeptides. *The MS1 peaks for the

monoisotopic m/z value were not visible and the measured m/z values for the second isotopes

are given instead.

14

D R G LT T R P G S G LT N I K T E E I S E V K M D A E F R H D S G Y E V H H Qc2 c3 c4 c18

z2z14 z4z16

c13

c2

z2

200 400 600 800 1000 1200 1400 1600 1800 2000m/z

915.436+

1098.505+

1373.364+

289.16

1294.593+

1139.012+

1431.652+

971.402+

848.362+

674.792+ 1714.782+459.26

268.12346.18c3

c4 z11

z14504.24z4

M

M

M

c18

c13

z+134

z161596.232+

c21

c21

z34

z+229

219.14

z29

m/z

1295.601295.26

1294.59

1294.941139.511139.01

1140.00

m/z

c13

z+134

400 800 1200 1600 2000

818.96+

Inte

nsity

818.39

m/z

m/z

MS2 915.436+

867.46+-25-15

A

B

915 916 916 917m/z

915.76915.93

916.09915.59 916.26

916.43915.43 916.60

916.76

Inte

nsity

Z=6

818.56

818.73818.89

819.06819.23

Z=6

Inte

nsity

Fig. S6. CID and ECD MS2 of Neu5AcHex(Neu5Ac)HexNAc-APP/Aβ–25-15 (A) MS1

(left) and CID MS2 (right) and (B) ECD MS2 spectrum of Neu5AcHex(Neu5Ac)HexNAc -

APP/Aβ–25-15. CID MS2 was acquired in ICR mode. Isotopic distributions of fragment ions

are demonstrated in the inserts supporting the evidence of significant fragments. Selected c-

and z-fragments are annotated and shown in relation to the peptide sequence. The attachment

site for the glycan was either Thr(–21) or Thr(–20).

15

Fig. S7. Identification of Thr (–21, –20 and –13) glycosylation sites. (A) ECD

spectrum of the –23-(–10) glycopeptide with three HexHexNAc glycans at Thr (–21,

–20 and –13). The amino acid numbering is according to the Aβ sequence. Ser(–16)

was excluded as a glycosylation site in this glycopeptide. Sialic acids were originally

present on the glycopeptides but were removed as a part of the enrichment protocol.

(B) CID MS2 spectrum of the same –23-(–10) glycopeptide as in (A). (C) CID MS2 of

the –23-(–10) glycopeptide with two HexHexNAc glycans. (D) CID MS2 of the –23-

(–10) glycopeptide with one HexHexNAc glycan. The peptide ion peak in the MS2

spectra (m/z 708.0) gave peptide fragmentation in the MS3 spectrum, which was used

to determine the peptide identity by Mascot database searching (See Table S2).

366.2

783.73+

608.33+

1415.8

675.83+

729.83+

662.33+

911.52+

890.62+

1073.62+

809.52+

1175.22+

992.12+

1618.0

1780.4

1821.2

400 600 800 1000 20001200 1400 18001600

Inte

nsity

A

–23-(–10)

890.52+

809.52+

992.32+

661.83+

608.33+ 1414.8

366.3204.0

400 600 800 1000 20001200 1400 18001600200

696.4

B –23-(–10)

D

708.02+

809.42+355.1

540.03+

366.1

204.2 577.3472.33+

200 400 600 800 1000 1200

Inte

nsity

–23-(–10)

358.22938.49

995.51

1082.55

1134.522+

1191.062+

1234.602+M - Ac

1234.603+

M1255.602+

1574.731430.68

1391.72

M837.743+

200 400 600 800 1000 1200 1400 1600 1800 2000

G L T T R P G S G L T N I Kz3z5z6z7z10z13

c7 c9 c12 c13

z3

z+15

z+17

z+16

z13

c13

c12

z10

c7

c9

CGLTTRPGSGLTNIKGLTTRPGSGLTNIK

GLTTRPGSGLTNIK

–23-(–10)

m/z

MS2 837.43+

MS2 715.73+

MS2 594.03+

m/z

m/z

Inte

nsity

16

366.2

863.92+

686.5557.3

770.92+

1373.3

965.42+

1170.3 1540.8

1726.9

872.42+

953.32+

1026.1y5y6

y14

y14

y14b11

b11

y14

400 600 800 1000 20001200 1400 18001600

–44-(–29)

CSVPANTENEVEPVDAR

y5y6y14

b11

568.43+

486.3 672.3

635.43+

850.52+

284.3

366.3204.1952.02+

400200 600 800 1000 1200

–9-5

569.32+

467.82+366.3204.1

934.5

600400200 800 1000 1200

–9-(–2)

A

B

TEEISEVKM*DAEFR

TEEISEVK

m/z

Inte

nsity

818.42+

522.3 322.2 1178.6

1227.9

1340.8

1378.6 1011.5 473.3

637.3

670.82+

735.32+

784.4

912.4

1200 1000 800 600 400

T E E I S E V K M D A E F Ry2

y2

y4

y4

y5

y5

y6

y6

y7

y7

y8

y8

y10

y10

y11

y11

b4

b4

y11

y12

y12

b10

b10

b12

b12

M -64

Inte

nsity

m/z

MS3 850.52+

MS2 689.03+

MS2 650.32+ MS2 1046.52+

Fig. S8. Identification of Thr (–39) and Ser(–5) / Thr(–9) glycosylation sites. (A)

CID MS2 of the –9-5 glycopeptide with one HexHexNAc glycan (left). The

glycosylation site was either Ser(–5) or Thr(–9). The deglycosylated peptide ion (m/z

850.5) present in the MS2 spectrum gave peptide fragmentation in the MS3 spectrum

(right), which was subjected to Mascot database searching to determine the

glycopeptide identity (see Table S2). *The methionine was oxidized (+16 Da)

resulting in a characteristic neutral loss of 64 Da from the oxidized Met in the CID

MS3 spectrum (M-64). (B) CID MS2 of the –9-(–2) glycopeptide carrying one

HexHexNAc glycan on either Ser(–5) or Thr(–9). (C) CID MS2 of the –44-(–29)

glycopeptide carrying one HexHexNAc glycan. The presence of glycosylated y14

peptide fragments in the MS2 spectrum pinpointed the glycosylation site to Thr(–39).

The y14 fragments represent the same fragmentation site as for the prominent y57

fragment ions for the APP/AβX-15 glycopeptides (see Fig. 4 and Fig. S5).

17

Mean intensity ratio of glycosylated form to unglycosylated form [%]

A!1-18

A!1-18

A!1-18

A!1-19

A!1-19

A!1-19

A!1-15Ac

A!1-17Ac

A!1-15

A!1-15

A!1-15

A!1-17

A!1-17

A!1-17

A!(–3-15)

A!4-17

A!4-17

A! 63-15)(–

A! 63-15)(–

A! 63-15)(–

A! 63-15)(–

A! 57-15)(–

A! 57-15)(–

A! 57-15)(–

A! 57-15)(–

A! 57-15)(–

A! 57-15)(–

A! 57-15)(–

A! 51-15)(–

A! 51-15)(–

A! 51-15)(–

A! 51-15)(–

A! 51-15)(–

A! 51-15)(–

A! 51-15)(–

A! 25-15)(–

A! 25-15)(–

0.1 1 10 100 1000 10000

non-AD AD

Fig. S9. Mean intensity ratios of APP/Aβ glycopeptides versus the corresponding

unglycosylated peptides for AD and non-AD patients. A mean ratio of 1 was set to

100% and the extensions of the bars represent one standard deviation around the

mean. The ratios were generally higher for AD compared to non-AD for most of the

Aβ1-X peptides, while the opposite was observed for most of the APP/Aβ X-15

peptides.

18

0.50 0.59 0.71 0.84 1.00 1.19 1.41 1.68 2.00

AD1AD2AD4AD3AD6AD5N3N1N7N5N2N4N6 790 770 730 670 570 520 510 490 410 390 360 340 200

[A!1-42] (pg/mL) / Sample

A! 63-15)(–

A! 63-15)(–

A! 63-15)(–

A! 63-15)(–

A! 63-15)(–

A! 58-15)(–

A! 57-15)(–

A! 57-15)(–

A! 57-15)(–

A! 57-15)(–

A! 57-15)(–

A! 57-15)(–

A! 57-15)(–

A! 57-15)(–

A! 51-15)(–

A! 51-15)(–

A! 51-15)(–

A! 51-15)(–

A! 51-15)(–

A! 51-15)(–

A! 51-15)(–

A! 51-15)(–

A! 47-15)(–

A! 45-15)(–

A! 36-15)(–

A! 25-15)(–

A! 25-15)(–

A! 25-15)(–

A! 22-15)(–

A! 21-15)(–

A! 14-15)(–

A! 11-15)(–

A! 05-15)(–

A!(–04-15)

Fig. S10. Heat map showing the relative signal intensities of individual

APP/AβX-15 peptides and glycopeptides for AD and non-AD patients. Data were

normalized in the same way as in Fig. 3 of the main text (see also Fig. S9 and Table

S1).

19

Table S1. Structures and relative intensities of 6E10 immunopurified APP/Aβ

peptides and glycopeptides of human CSF samples. The glycosylated/unglycosylated

spectral intensity ratios for Aβ peptides and glycopeptides from AD versus non-AD

patients (nonAD) are shown.

Peptide/Glycopeptidea Calculated

monoisotopicmass (Da)

MS2 datab AD vs. C (mean C set to 1)c Mean

intensity (log10)d

Mean nonAD

Std dev. non-AD

Mean AD

Std dev. AD

Αβ1-13 1560.6593 Y 1.0 0.2 0.7 0.2 5.3

Αβ1-14 1697.7182 Y 1.0 0.1 0.9 0.1 5.9

Αβ1-15 1825.7768 S4G 1.0 0.2 0.9 0.2 6.0 SaHHn-Αβ1-15 2482.0044 S3A 1.0 0.3 1.1 0.5 3.6

Sa2HHn-Αβ1-15 2773.0999 F1A, F2A 1.0 0.1 1.4 0.1 5.1 LaSa2SaHHn-Αβ1-15 3046.1847 F1D 1.0 0.2 1.9 0.1 4.4

Sa3HHn-Αβ1-15 3064.1953 F1B, S4E 1.0 0.1 1.6 0.0 4.7 Ac-Sa3HHn-Αβ1-15 3106.2058 F1C 1.0 0.3 2.3 0.3 4.0

Αβ1-16 1953.8718 Y 1.0 0.2 1.1 0.2 5.3 LaSa2SaHHn-Αβ1-16 3174.2797 S3E Below threshold <3.0

Sa3HHn-Αβ1-16 3192.2902 S3D Below threshold <3.0

Αβ1-17 2066.9559 Y 1.0 0.1 1.0 0.1 7.3 SaHHn-Αβ1-17 2723.1835 S3G 1.0 0.4 1.1 0.3 4.6

Sa2HHn-Αβ1-17 3014.2789 F2B, S3H 1.0 0.1 1.2 0.1 5.8 LaSa2SaHHn-Αβ1-17 3287.3637 S3K 1.0 0.2 1.6 0.1 4.5

Sa3HHn-Αβ1-17 3305.3743 S3I, S4F 1.0 0.1 1.2 0.3 4.7 Ac-Sa3HHn-Αβ1-17 3347.3849 S3J 1.0 0.2 1.5 0.1 4.1

Αβ1-18 2166.0243 Y 1.0 0.2 1.0 0.1 6.0 Sa2HHn-Αβ1-18 3113.3473 S3O 1.0 0.1 1.1 0.1 4.4

LaSa2SaHHn-Αβ1-18 3386.4321 S3R 1.0 0.3 2.5 0.5 4.1 Sa3HHn-Αβ1-18 3404.4427 S3P 1.0 0.2 1.4 0.4 4.5

Ac-Sa3HHn-Αβ1-18 3446.4533 S3Q 1.0 0.2 1.3 0.5 3.2

Αβ1-19 2313.0927 S4K 1.0 0.1 1.2 0.1 7.3 Sa2HHn-Αβ1-19 3260.4157 S3S 1.0 0.1 1.4 0.2 5.3

LaSa2SaHHn-Αβ1-19 3533.5006 S3V 1.0 0.4 2.5 0.6 5.2 Sa3HHn-Αβ1-19 3551.5111 S3T, S4J 1.0 0.2 1.9 0.4 5.4

Ac-Sa3HHn-Αβ1-19 3593.5217 S3U 1.0 0.2 1.4 0.3 4.2

Αβ1-20 2460.1611 Y 1.0 0.2 1.0 0.2 5.9 Sa2HHn-Αβ1-20 3407.4841 S3W 1.0 0.5 0.8 0.4 3.9

Αβ1-28 3260.5275 Y 1.0 0.3 0.5 0.2 6.3 Αβ1-30 3388.5861 Y 1.0 0.1 1.1 0.1 6.7 Αβ1-33 3671.7757 Y 1.0 0.1 1.0 0.1 7.5 Αβ1-34 3784.8598 Y 1.0 0.1 0.8 0.1 7.6 Αβ1-37 4071.9901 Y 1.0 0.0 1.0 0.0 7.9 Αβ1-38 4129.0116 Y 1.0 0.0 1.0 0.0 8.6 Αβ1-39 4228.0800 Y 1.0 0.0 1.2 0.1 7.8 Αβ1-40 4327.1484 Y 1.0 0.0 1.0 0.0 8.8

20

Αβ1-42 4511.2696 Y 1.0 0.2 0.5 0.1 6.6

Αβ4-15 1510.6702 N Below threshold <3.0 Sa2HHn-Αβ4-15 2457.9932 S3B 1.0 0.4 2.2 0.9 4.0

LaSa2SaHHn-Αβ4-15 2731.0781 S3C Below threshold <3.0

Αβ4-17 1751.8492 N 1.0 0.2 0.9 0.1 4.3 Sa2HHn-Αβ4-17 2699.1722 S3L, S4H 1.0 0.1 1.4 0.1 5.3 Sa3HHn-Αβ4-17 2990.2676 S3M 1.0 0.1 2.1 0.1 4.1

Αβ5-17 1604.7808 N 1.0 0.2 0.6 0.1 4.3 Sa2HHn-Αβ5-17 2552.1038 S3N Below threshold <3.0

Αβ(–3-15) 2183.9812 N 1.0 0.4 0.4 0.2 3.6 Sa2HHn-Αβ(–3-15) 3131.3043 S3F, S4I 1.0 0.1 1.1 0.2 4.4

Αβ(–4-15) 2313.0238 Y 1.0 0.2 0.7 0.1 5.2

Αβ(–5-15) 2400.0558 Y 1.0 0.1 1.0 0.1 5.5

Αβ(–11-15) 3113.4518 Y 1.0 0.2 1.0 0.1 5.9

Αβ(–14-15) 3441.6264 Y 1.0 0.2 1.0 0.2 5.5

Αβ(–21-15) 4097.9506 Y 1.0 0.2 1.1 0.1 6.0

Αβ(–22-15) 4211.0347 Y 1.0 0.2 1.4 0.2 6.4

Αβ(–25-15) 4539.1842 Y 1.0 0.1 1.0 0.1 7.3 SaHHn-Αβ(–25-15) 5195.4118 N 1.0 0.1 1.0 0.2 6.3

Sa2HHn-Αβ(–25-15) 5486.5073 S6 1.0 0.3 1.4 0.3 6.8 (SaHHn)3-Αβ(–25-15) 6507.8671 F4B 1.0 0.2 0.7 0.2 5.8

Αβ(–45-15) 6601.1445 Y 1.0 0.2 1.3 0.2 7.0 Sa2HHn-Αβ(–45-15) 7548.4676 S5A 1.0 0.1 0.9 0.1 6.6

Αβ(–51-15) 7286.4418 Y 1.0 0.2 1.0 0.1 6.4 SaHHn-Αβ(–51-15) 7942.6694 S5C 1.0 0.1 0.9 0.2 7.1

Sa2HHn-Αβ(–51-15) 8233.7649 S5D 1.0 0.1 0.9 0.3 6.4 (SaHHn)2-Αβ(–51-15) 8598.8970 S5E 1.0 0.1 0.5 0.2 6.5

Sa2HHnSaHHn-Αβ(–51-15) 8889.9925 N 1.0 0.4 1.0 0.5 6.0 (SaHHn)3-Αβ(–51-15) 9255.1247 F4C, S5B 1.0 0.2 0.5 0.1 7.1

Sa2HHn(SaHHn)2-Αβ(–51-15) 9546.2201 S5F 1.0 0.2 0.7 0.0 6.7 (SaHHn)4-Αβ(–51-15) 9911.3523 S5G 1.0 0.5 1.1 0.6 6.8

Sa2HHn(SaHHn)3-Αβ(–51-15) 10202.4477 Y 1.0 0.4 1.1 0.3 6.6 (Sa2HHn)2(SaHHn)2-Αβ(–51-15) 10493.5431 N 1.0 0.3 0.8 0.2 6.0

(SaHHn)5-Αβ(–51-15) 10567.5799 N 1.0 0.4 0.7 0.3 5.8 Sa2HHn(SaHHn)4-Αβ(–51-15) 10858.6753 N 1.0 0.3 0.6 0.1 5.3

Αβ(–57-15) 7967.7751 Y 1.0 0.1 1.0 0.1 7.2 SaHHn-Αβ(–57-15) 8624.0027 S5H 1.0 0.3 0.8 0.3 6.6

Sa2HHn-Αβ(–57-15) 8915.0982 S5I 1.0 0.1 0.8 0.2 6.8 (SaHHn)2-Αβ(–57-15) 9280.2303 N 1.0 0.3 0.9 0.2 6.4

Sa2HHnSaHHn-Αβ(–57-15) 9571.3258 N 1.0 0.2 0.8 0.1 5.8 (SaHHn)3-Αβ(–57-15) 9936.4580 F4D 1.0 0.1 0.7 0.1 6.9

Sa2HHn(SaHHn)2-Αβ(–57-15) 10227.5534 N 1.0 0.2 0.8 0.1 6.5 (SaHHn)4-Αβ(–57-15) 10592.6856 S5J 1.0 0.3 0.6 0.2 6.9

Sa2HHn(SaHHn)3-Αβ(–57-15) 10883.7810 N 1.0 0.3 0.7 0.2 6.6 (Sa2HHn)2(SaHHn)2-Αβ(–57-15) 11174.8764 N 1.0 0.5 0.5 0.3 5.6

(SaHHn)5-Αβ(–57-15) 11248.9132 N 1.0 0.2 75.7 40.0 6.2

21

Sa2HHn(SaHHn)4-Αβ(–57-15) 11540.0086 N 1.0 0.3 0.9 0.5 4.8

Αβ(–58-15) 8054.8072 Y 1.0 0.2 1.2 0.1 6.7

Αβ(–63-15) 8601.0510 N 1.0 0.1 1.0 0.1 6.4 SaHHn-Αβ(–63-15) 9257.2786 N 1.0 0.3 1.0 0.3 6.7

Sa2HHn-Αβ(–63-15) 9548.3741 N 1.0 0.5 1.3 0.6 6.2 (SaHHn)2-Αβ(–63-15) 9913.5062 N 1.0 0.6 36.2 17.5 6.3

Sa2HHnSaHHn-Αβ(–63-15) 10204.6017 N 1.0 0.3 0.9 0.1 5.9 (SaHHn)3-Αβ(–63-15) 10569.7339 S5K 1.0 0.1 0.7 0.1 7.0

Sa2HHn(SaHHn)2-Αβ(–63-15) 10860.8293 N 1.0 0.3 0.8 0.1 6.6 (SaHHn)4-Αβ(–63-15) 11225.9615 S5L 1.0 0.3 0.5 0.1 7.0

Sa2HHn(SaHHn)3-Αβ(–63-15) 11517.0569 F4E 1.0 0.4 0.9 0.2 6.8 (Sa2HHn)2(SaHHn)2-Αβ(–63-15) 11808.1523 N 1.0 0.3 1.0 0.4 6.1

(SaHHn)5-Αβ(–63-15) 11882.1891 N 1.0 0.4 0.4 0.3 6.0 Sa2HHn(SaHHn)4-Αβ(–63-15) 12173.2845 N 1.0 0.3 0.8 0.2 5.7

a Sa = Neu5Ac, H = Hex, Hn = HexNAc, LaSa2 = two Neu5Ac linked with lactone

formation, Ac = O-acetyl b Y = CID MS2 data obtained but not shown in this publication; N = MS2 data not

obtained; F# = MS2 data obtained and shown in Fig. #; S# = MS2 data obtained and

shown in Fig. S# c Values obtained as described in the Materials and Methods section. Below threshold

= no signal detected in the AD vs. non-AD study, but detected in other experiments. d log10 values of mean peak intensity of the 13 spectra used in the quantitative AD vs.

non-AD

study. Signal threshold was subjectively set to 1000 (log101000 = 3). Peptides with

log10 peak intensity <3.0 in this set were thus below the detection threshold.

Table S2. Sequence and structures of APP/Aβ glycopeptides obtained after sialic acid

capture-and-release of peptides immunopurified from CSF with the 6E10 antibody.

CID MS2 spectra of these glycopeptides are shown in Figs. S7 and S8.

Peptide APP/Aβ region Glycan Mascot

score Threshold (p<0.05)

K.TEEISEVK.M –9-(–2) HexHexNAc 34 >25 K.TEEISEVKMDAEFR.H –9 - 5 HexHexNAc 62 >21 R.GLTTRPGSGLTNIK.T –23-(–10) (HexHexNAc)3 - - R.GLTTRPGSGLTNIK.T –23-(–10) (HexHexNAc)2 19 >18 R.GLTTRPGSGLTNIK.T –23-(–10) HexHexNAc 39 >19 D.SVPANTENEVEPVDAR.P –44-(–29) HexHexNAc 43 >25