Novel O-linked Sialoglycan Structures in Human Urinary ... · S-1 SUPPORTING INFORMATION for the article “Novel O-linked Sialoglycan Structures in Human Urinary Glycoproteins”

S-1 SUPPORTING INFORMATION for the article

“Novel O-linked Sialoglycan Structures in Human Urinary Glycoproteins”

A. Pap, E. Tasnadi, K.F. Medzihradszky and Z. Darula

Table of Contents

- Cover page

- Table S-1 - Novel glycans S-2

- Internal & non-reducing end glycan fragments (calculated masses) S-5

- Legends and Annotations S-6

- Manually annotated HCD and EThcD spectra of different glycoforms of AVAVTLQSH S-7 and DFTAAFPR peptides, and schemes with alternative structures, when there is any, pointing out the fragmentation differences

The most likely structure of new glycoforms was deciphered manually, using both HCD and EThcD data. Obviously, the identity of the sugar units and their linkage positions cannot be assigned from these data. However, this supplement demonstrates that EThcD spectra acquired with 15% NCE may deliver structural information on the glycan part of a glycopeptide. All AVAVTLQSH spectra presented here have proof for the site assignment (modified c7 and/or unmodified z4 peptide fragments).

Electronic Supplementary Material (ESI) for Molecular Omics.This journal is © The Royal Society of Chemistry 2020

S-2

Monoisotopic additive mass

698.2382 1167.4177 1224.4392 1370.4971

Composition NHAAc N2H2FA N3H2A N3H2FA

Structure

Ac


1386.4920 1392.4120 1645.5612 1677.5874 1687.5718

Composition N3H3A N2H2A2Sulfo N2H2A2AAc N3H3A2 N2H2AAAc2

Structure

SO3H

Ac Ac

Ac

This isomer has been reported in [Darula, Z.; Pap A.; Medzihradszky, K. F. J Proteome Res. 2019, 18, 280-291.] Table S-1 Novel glycans

N: HexNAc, H: Hex, F: Fuc, A: NeuAc, AAc: NeuAcAc

S-3


1735.6293 1751.6242 1969.7032 2042.7196 2116.7564

Composition N4H3FA N4H4A N3H3F2A2 N4H4A2 N5H5A

Structure

Table S-1 Novel glycans (contd.)

N: HexNAc, H: Hex, F: Fuc, A: NeuAc

S-4


2333.8150 2391.8569 2407.8518

Composition N4H4A3 N5H4FA2 N5H5A2

Structure

Table S-1 Novel glycans (contd.)

N: HexNAc, H: Hex, F: Fuc, A: NeuAc

S-5 Internal & non-reducing end glycan fragments (calculated masses)

m/z Glycan composition

186.0761 HexNAc-H2O

204.0867 HexNAc

274.0921 NeuAc-H2O

292.1027 NeuAc

314.0846 NeuAc+Na

316.1027 NeuAcAc-H2O

334.1133 NeuAcAc

366.1395 HexNAcHex

388.1214 HexNAcHex+Na

407.1661 HexNAc2

454.1555 HexNeuAc

476.1374 HexNeuAc+Na

495.1821 HexNAcNeuAc

512.1974 HexNAcHexFuc

528.1923 HexNAcHex2

m/z Glycan composition

569.2189 HexNAc2Hex

657.2349 HexNAcHexNeuAc

698.2615 HexNAc2NeuAc

699.2455 HexNAcHexNeuAcAc

715.2768 HexNAc2HexFuc

731.2717 HexNAc2Hex2

803.2928 HexNAcHexFucNeuAc

1022.3671 HexNAc2Hex2NeuAc

1096.4039 HexNAc3Hex3

1168.4250 HexNAc2Hex2FucNeuAc

1387.4993 HexNAc3Hex3NeuAc

1371.5044 HexNAc3Hex2FucNeuAc

1678.5947 HexNAc3Hex3NeuAc2

S-6 Legends and Annotations

In Figures S-1-14 HCD spectra are presented in the upper panels, EThcD data are shown in the lower panels. Peptide fragments are labeled

according to the Biemann nomenclature [Biemann K. Nomenclature for peptide fragment ions (positive ions). Methods Enzymol. 1990, 193,

886-887.]. In most cases the peptide fragments are rather weak and the HCD data may reveal the presence of coeluting/cofragmenting

components. Glycan fragments are labeled according to the Domon-Costello nomenclature [Domon, B.; Costello, C. E. Glycoconjugate J.

1988, 5, 397-409.], and the cleavages are indicated in the schemes included. Internal glycan fragments are labeled with cartoons, according

to the CFG recommendations. In the EThcD spectra the precursor and its charge reduced forms are indicated as „pr(charge)”. The charge

reduced form of coeluting molecules are labeled with „Coel.(charge)”. When a fragment is formed via multiple glycosidic bond cleavages

than the following label format is used: Y2,3. When a particular mass may represent different structures, the following format is used:

Y2|Y2.

Figure S-15 – displays the EThcD data of isomeric blood-type A antigens, Type 3 (upper panel), Type 1 or 2 (lower panel).

As we reported earlier [Darula, Z.; Pap, Á.; Medzihradszky, K. F. J Proteome Res. 2019, 18, 280–291.], in some of the spectra we observed

fragments that indicated sialic acid or fucose migration, a known phenomenon in glycan analysis [Wuhrer, M.; Deelder, A. M.; van der Burgt,

Y. E. Mass Spectrom Rev. 2011, 30, 664-680.]. Such fragments are clearly annotated.

D:\PA\...\180417_06 4/17/2018 8:05:27 PM 180327_03_GlcNAc

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Time (min)

180417_06 #12926 RT: 42.59 AV: 1 NL: 8.92E5T: FTMS + c NSI d Full ms2 [email protected] [100.0000-1635.0000]

100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600

m/z

0

10

20

30

40

50

60

70

80

90

100

Re

lative

Ab

un

da

nce

925.512

316.103

204.087

1128.591186.076

274.093 811.397334.114

585.297366.140 755.411 1290.628907.498238.072 484.258 683.413 1208.538442.269 964.438555.353 612.245 1421.6861092.5061043.474

180417_06 #12928 RT: 42.60 AV: 1 NL: 4.06E5T: FTMS + c NSI d sa Full ms2 [email protected] [email protected] [150.0000-2000.0000]

200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000

m/z

0

10

20

30

40

50

60

70

80

90

100

Re

lative

Ab

un

da

nce

1624.757

812.379

1290.6451607.736

1128.589 1267.521925.512 1564.732334.114 1437.6311367.591645.826564.799 699.248496.167 1715.832408.706204.087

Y0 Y1

Y2

pr(2+)

pr(+)

1366.585 z6 z7

Y1(2+)

Y2(2+) z5

1397.661 1398.683

c-17

c7

1485.698 c8

1536.669 (15.6 ppm)

z8

Y0

Y1 Y0+HexNAcHex

y4 y5 y7

684.375 (10 ppm)

y6

b5

171.114 -> b2 156.077 -> y1

242.150 -> b3 243.111 -> y2

341.222 -> b4 371.170 -> y3 770.441 -> b8

b6

b7

B2

Y2

Ac

Y0

Y1

B1

B3

B3 B2 B1

B1

S-7 Figure S-1 812.3802 (2+) → AVAVT(698.2382)LQSH2+

D:\PA\...\180420_05 4/20/2018 6:28:18 PM 180327_01_Shoulder

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Time (min)


100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000

m/z

0

10

20

30

40

50

60

70

80

90

100

Re

lative

Ab

un

da

nce

925.509

204.086

138.055274.092

366.1391128.588

292.1021290.639585.297 907.501484.250 770.439314.084 683.407 1047.440967.518407.168 1494.710

1087.557

1412.9491193.528


200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000

m/z

0

10

20

30

40

50

60

70

80

90

100

Re

lative

Ab

un

da

nce

1046.966

1801.836

1954.886

1906.808973.940901.419

1835.769

1570.1141290.645 1736.704803.296512.198366.139 1436.692657.232 1168.419 1655.767292.102 747.367178.233 1334.830454.151

1581.737

Y1

1128.590

Y1,1

*

*: 1784.835 -> Y2

Y2,1

Y2 Y2-Fuc

c8

c7 1867.851

z7

z6

z5

y4 y5

-Fuc(2+) Y2(2+)

Y2,2|Y1-Fuc (2+)

Fucose migration Pr+HexNAcHexFuc

Coel. (2+)

Y0

Y1

Y0+HexNAcHex

Y0+HexNAc2

1331.659

Y0+HexNAc2Hex 1493.722

b8 b7 Y0-H2O

242.150 -> b3 156.076 -> y1

341.218 -> b4 243.110 -> y2

442.264 -> b5 371.167 -> y3 555.348 -> b6 684.373 -> y6

755.406 -> y7

B2 B1

B2

B2 B2

B1

Y1 Y1

Y2 Y2

B3

Y0

B3

925.510

Y0

**

**: NeuAc migration:

pr(2+)


D:\PA\...\180417_13 4/18/2018 7:16:55 AM 180327_04_GlcNAc

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95

Time (min)


100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000

m/z

0

10

20

30

40

50

60

70

80

90

100

Re

lative

Ab

un

da

nce

204.087

925.513

717.403

274.093

126.055

186.077

366.141 1128.595699.241

488.235292.102 406.1731290.649

1494.731312.086 550.180426.132 1331.682783.385657.235 907.498 1178.6451084.530


200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000

m/z

0

10

20

30

40

50

60

70

80

90

100

Re

lative

Ab

un

da

nce

717.267

1054.978

929.935

204.087 1433.625973.943 1858.8771683.740657.234274.093 468.233

1032.462

893.420747.365366.143 1796.809 1964.841848.909 1120.492 1290.619584.336

892.912(2+)

z4

355.151 z3

Y2

(2+)

-HexNAc(*)

(2+)

Y2(2+)

-Ac

pr(2+) Y1

(2+)

Y1

1031.949 z8

2+

c5 c6 Y3

1892.794 z6

c-17

c7 1924.889

z+17

Y0

Y1

Y0+HexNAcHex

Y0+HexNAc2

1493.727

Y0+HexNAc2Hex

Y0-H2O

242.151 -> b2 y1 -> 156.077 683.414 -> b7 y4 -> 484.252 770.452 -> b8 y5 -> 585.297 y7 -> 755.410

Coel. (+)

w4: 425.179 w3: 297.120

B2

B1

1128.595 Y1,1

pr(3+)

292.103


Y2

Y1 Y1

* B2

S-10

The most likely structure

366 892 (2+)

HexNAc loss from the precursor

Potential structures for the HexNAc3Hex2NeuAc (1224.4392) composition:

Y1β B3β

These fragment ions were not detected

1290

860

The other option

S-11 D:\PA\...\180420_19_i 180410_01_GlcNAc

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95

Time (min)

180420_19_i #15300 RT: 43.98 AV: 1 NL: 8.43E5T: FTMS + c NSI d Full ms2 [email protected] [100.0000-2000.0000]

100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000

m/z

0

10

20

30

40

50

60

70

80

90

100

Re

lative

Ab

un

da

nce

204.086

1127.532

366.139274.092

924.455

1289.587

1492.667

771.346292.102 1330.613

632.773536.223 1654.7141172.548 1580.689326.170 1375.633431.192 649.313 988.452

753.354

380.146

903.400

1273.617

1065.375809.433

1476.649

180420_19_i #15302 RT: 43.99 AV: 1 NL: 3.92E5T: FTMS + c NSI d sa Full ms2 [email protected] [email protected] [150.0000-2000.0000]

200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000

m/z

0

10

20

30

40

50

60

70

80

90

100

Re

lative

Ab

un

da

nce

771.344

1135.475

1156.484

1541.6551010.929

366.139

929.904731.2731767.676 1909.756403.220

1483.633 1564.6961104.506545.296204.086 274.092 474.259 643.276 1946.830790.846 855.368 1029.206

Figure S-4 770.9880(3+) → DFT(1386.492)AAFPR3+

Y0

Y1

Y0+HexNAcHex

Y0 Y1

Y0+HexNAc2

Y0+HexNAc2Hex

Y0+HexNAcHexNeuAc

Y0+HexNAc2Hex2

y1 -> 175.118 y2 -> 272.171 y3 -> 419.240 y5 -> 561.312 y6 -> 809.433

z3 z4 z5

B4 Y1

(2+)

-Hex2NeuAc 848.383(2+)

-HexNeuAc 929.406(2+)

-HexHexNAc 973.414(2+)

-NeuAc 1010.430(2+)

-Ac 1134.979(2+)

Pr(2+) 1155.995(2+)

Coel. (+)

c3

c4 1838.727

c5

-HexHexNAc 1945.787

Y1

B4

Pr(3+)

F:\Adam\...\180417_05 04/17/18 18:09:13 180327_03_GlcNAc

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95

Time (min)


100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000

m/z

0

10

20

30

40

50

60

70

80

90

100

Re

lative

Ab

un

da

nce

274.093

204.087

366.1411128.575

138.055

292.1041331.656

728.313455.263 1493.705925.515 1423.602550.287 815.329626.773 1099.571407.169355.068 1290.6271220.523970.505 1785.8081535.687 1696.7601406.573

1155.538


200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000

m/z

0

10

20

30

40

50

60

70

80

90

100

Re

lative

Ab

un

da

nce

814.340

1222.021

1075.975

1200.520

1627.687

292.104 892.906994.447657.238366.141 1610.669

1365.5741162.988204.087 538.313 731.273 1039.456 1497.606 1904.7621785.795930.421

779.677

454.156 848.900 1712.767

246.145

1667.733

1312.457

1668.728

1959.837597.165

pr (2+) pr (3+)

Coel. (+)

B1α|B1

B2

B2α|B3

*

*: 468.234 -> z4

**: Y2,3(2+)

Y2,2

(2+) Y1|Y1

(2+)

**

Y2

(2+)

Y2|Y3 1075.474(2+)

-Ac(2+)

1784.808

Y1|Y1

Y1

Y0

Y0+HexNAc2

1534.708 (-26ppm)

Y0+HexNAc3

Y0+HexNAc3Hex

1784.793 (-13ppm)

Y0+HexNAc2HexNeuAc

Y0+HexNAc2Hex It’s a mixture. No peptide fragments to ID.


1119.475 (2+)

-HexNAc

Y2

Y3

Y2

Y1 Y1

B3 B2α

B2

B1

B1α

S-13

The most likely structure

892 (2+) 657

HexNAc loss from the precursor

Potential other structures for the HexNAc3Hex2NeuAc2 (1515.5346) composition:

Other options

Y1β

B3β 860

791(2+)

Was not detected The HCD fragment that could prove this structure (peptide+HexNAc3) is outside of the acceptable mass accuracy range.

D:\PA\...\180417_26 04/18/18 20:24:29 180410_03_GlcNAc

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100

Time (min)


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m/z

0

10

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30

40

50

60

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Re

lative

Ab

un

da

nce

274.093

204.087925.512

366.140

138.055867.433

1128.592

292.103

1290.644

1493.728569.220 1581.7401331.664407.169 657.235 850.277731.266 1655.799495.186314.083 1213.582 1386.057 1947.867

1167.614

1087.5591010.987


200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000

m/z

0

10

20

30

40

50

60

70

80

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Re

lative

Ab

un

da

nce

867.4351303.062

1281.554

1157.005

1022.369973.942 1736.7171075.986292.103

657.235468.233366.142 791.371228.098 1258.526 1566.717 1719.720731.272 1447.603 1947.869930.431

1142.502

828.906

1192.533

409.037 495.187 569.224

z2*

355.150 z3

z4 B3

B1|B1

Y1

(2+)

89

2.9

13

(2

+) Y2

pr (3+)

Y3

(2+) B5

B1α

B1

B3

Y1

Y2

Y3

B5 Y1α

Y4

1075.485 (2+)

Y1|Y4

1156.515 (2+)

Y2|Y5 -Ac (2+)

pr (2+)

z82+

*

*:1233.044 (2+) -> c82+

Coel. (+)

Y2α

Y5

Y3 1946.867

**

**: 1581.737 -> Y1

Y0

Y1

Y0+HexNAcHex

Y0+HexNAc2

Y0+HexNAc2Hex

Y0+HexNAc2Hex2

Y0+HexNAcHexNeuAc

B3

683.413 -> b7 585.300 -> y5 770.439 -> b8 755.398 -> y7


S-15 D:\PA\...\180417_07 04/17/18 22:01:38 180327_03_Shoulder

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Time (min)


100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000

m/z

0

10

20

30

40

50

60

70

80

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100

Re

lative

Ab

un

da

nce

871.460

204.087

925.513

274.093

366.140138.055

1128.594

1084.546 1290.650

1053.457619.312 676.656 1205.505407.163 1331.683 1493.726476.138 853.417 1623.765985.396 1434.620775.099


200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000

m/z

0

10

20

30

40

50

60

70

80

90

100

Re

lative

Ab

un

da

nce

871.436

1308.054

1140.499 1742.7621286.547

994.948

1726.717

334.113

204.087 812.387292.105

1161.519 1628.6401450.679657.235 790.823 1084.471

468.2361269.550

913.9221989.916

409.978 974.432

x5 x5

Y1

Y4

Y3

Y2

B1

B1

B4

Ac

Y2

Y1

Ac

B1|B1

z4

z2*

228.099

●

366.140

Y1 (2+)

Y2

(2+)

990.355

B4

Y3

(2+) Y2|Y4

(2+) ●●

●: 355.149 -> z3

●●: 973.939(2+) -> Y1,1

1059.460(2+)

Y1

1286.043(2+) -Ac

-NeuAc(2+)

Coel. (+) 1307.056 pr(2+)

AVAVT(1687.5718)LQSH

Y0

Y1

Y0+HexNAcHex

Y0+HexNAc2

Y0+HexNAc2Hex

156.077 -> y1 442.269 -> b5

371.168 -> y3 555.355 -> b6

484.253 -> y4 683.407 -> b7

585.304 -> y5 770.443 -> b8

755.407 -> y7

It’s a mixture.

Figure S-7 871.7020 (3+) → AVAVT(1687.5718)LQSH3+

S-16 Another potential structure for the HexNAc4Hex3FucNeuAc (1735.6293) composition: (The most likely structure and the supporting EThcD spectrum can be found in the article as Figure 2.)

Y1

892.5 (2+)

B2 877

Y1

718.5 (2+)

B2 1225

S-17 D:\PA\...\180420_19_i 180410_01_GlcNAc

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95

Time (min)

180420_19_i #14668 RT: 42.64 AV: 1 NL: 1.08E6T: FTMS + c NSI d Full ms2 [email protected] [100.0000-2000.0000]

100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000

m/z

0

10

20

30

40

50

60

70

80

90

100

Re

lative

Ab

un

da

nce

204.086

138.055

366.139

1289.586

274.092 893.4741127.534

1492.668 1654.718

1330.611292.1021580.679528.193 1858.792731.276 875.448

215.054

1165.053407.167 684.333

1536.688

1945.809968.453 1062.490 1783.7571696.766

1375.625

1267.051

343.196

180420_19_i #14670 RT: 42.64 AV: 1 NL: 4.47E5T: FTMS + c NSI d sa Full ms2 [email protected] [email protected] [150.0000-2000.0000]

200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000

m/z

0

10

20

30

40

50

60

70

80

90

100

Re

lative

Ab

un

da

nce

893.335

1339.574

1156.476366.139

1193.494

1785.773973.410731.2711096.408204.087 545.293403.221 474.259 690.246 1946.8191767.7711250.018292.102 1581.6901010.442 1654.6891493.654790.849 924.454

348.797

892.6997(3+) → DFT(1751.6242)AAFPR3+ Figure S-8

924.455

Y0

Y1

Y0+HexNAcHex

Y0+HexNAc2

Y0+HexNAc2Hex

Y0+HexNAcHexNeuAc

Y0+HexNAc2Hex2 Y0+HexNAc2HexNeuAc

Y0+HexNAc3Hex2

*

*: Y0+HexNAc2Hex2NeuAc

z3 z4 z5

B4 Y1

(2+)

Pr(3+)

Y0

Y3

(2+)

-NeuAc -HexNAcHex

(2+)

B6

1155.984 (2+)

1193.004 (2+)

-NeuAc

*

*: 1317.547(2+) → -Ac

1338.582 (2+) Pr(2+)

Coel.(+) 1945.811

Y3

Y1

B6

Y3

B4 Y5

Y5

D:\PA\...\180417_07 04/17/18 22:01:38 180327_03_Shoulder

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95

Time (min)


100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000

m/z

0

10

20

30

40

50

60

70

80

90

100

Re

lative

Ab

un

da

nce

274.093

204.087

925.512

366.140

168.066 292.103

1128.592

966.4211290.650

512.197 1493.723569.221 657.234 1241.545 1581.737821.346771.020 1035.556 1331.680407.168 1655.774

1147.4311086.745868.670

1728.796 1802.810


200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000

m/z

0

10

20

30

40

50

60

70

80

90

100

Re

lative

Ab

un

da

nce

1449.106

966.414

1427.599

1303.061

1931.725

292.103 1230.0371046.970274.093 657.233 1914.803791.378468.233366.140 1157.003

1287.797

1379.577 1639.734864.403

948.334

1774.127204.088

355.148

z3

z4

803.297

B3

B3-Fuc

Y1

(2+)

973.945(2+) Y3-Fuc

B1|B1 Y3

(2+)

973.945(2+) (Y1|Y4)-2Fuc

*

*:1075.472(2+) -> Y4-2Fuc :1148.515(2+)-> Y4-Fuc

●:1168.433 -> B5-Fuc

1156.505(2+)

(Y2|Y5)-2Fuc

●

1229.539(2+)

(Y2|Y5)-Fuc

1302.038(2+) -2Fuc

-Ac(2+)

pr(2+)

Coel. (+)

Y0

Y1

Y0+HexNAcHex

Y0+HexNAc2

Y0+HexNAc2Hex

Y0+HexNAcHexNeuAc

Y0+HexNAc2Hex2

1436.688

Y0+HexNAcHexFuc

*

*:1639.788 -> Y0+HexNAc2HexFuc

B3-Fuc

Y4

Y5

Y3

Y1 Y1

Y2

B1

B2

B1

B2

B3

B5

454.155

B2|B2

pr(3+)


D:\PA\...\180417_26 4/18/2018 8:24:29 PM 180410_03_GlcNAc

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100

Time (min)


100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000

m/z

0

10

20

30

40

50

60

70

80

90

100

Re

lative

Ab

un

da

nce

204.087

274.093

366.140

138.055

925.512

699.342292.103

1128.594990.508 1290.648569.219 1493.731657.236 731.273407.167 1214.575 1581.739 1655.781495.181 796.379 1331.662316.103 1741.741 1903.8031011.480863.088 1417.658


200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000

m/z

0

10

20

30

40

50

60

70

80

90

100

Re

lative

Ab

un

da

nce

1485.623

1464.115

989.457

1339.577

1978.8121156.521657.236292.103 1258.550366.142 1134.965 1383.041974.442791.376 1022.371

1306.210

204.087 1962.762893.407698.265410.308 1222.524 1651.040454.158 1833.779

1447.601

341.254

pr(2+)

-Ac (2+)

Y2|Y7

** *

**:1387.508(2+)-> B7

*:468.238 -> z4

973.940 (2+)

Y0

Y1

Y0+HexNAcHex

Y0+HexNAc2Hex

Y0+HexNAc2Hex2

Y1|Y6

Y5

(2+) Y3 Y1

(2+)

Sialic acid migration

(3+)

B5 B3

1258.038 (2+)

1339.075 (2+) pr(3+)

Coel. (+)

B2|B2

B1|B1

699.009

Y1

Y3

Y5

Y6

Y7

Y1

Y2

B5

B7

B1

B2

B3

B1

B2

Y0+HexNAc2

Y0+HexNAcHexNeuAc

B3

It’s a mixture.

(3+)

770.442 -> b8

796.035


S-20 D:\PA\...\180417_13 04/18/18 07:16:55 180327_04_GlcNAc

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95

Time (min)


100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000

m/z

0

10

20

30

40

50

60

70

80

90

100

Re

lative

Ab

un

da

nce

204.087366.141

138.055

274.093

1014.449 1289.595292.104 1654.728

1492.672924.456 1127.535731.273569.218 1209.562785.349 1580.693414.237 481.265 657.233 1857.797950.402 1945.7991108.519 1330.612883.368 1783.763314.0861695.791


200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000

m/z

0

10

20

30

40

50

60

70

80

90

100

Re

lative

Ab

un

da

nce

1014.453

1500.605

1339.048366.142

973.425 1096.411403.221 731.273 1155.966545.294204.086 923.429 1983.852292.104 1254.174 1689.651 1880.8321432.582657.232474.257

1014.4027 (3+) → DFT(2116.7564)AAFPR3+ Figure S-11

Y0 Y1

Y0+HexNAcHex

Y0+HexNAc2

Y0+HexNAc2Hex

Y0+HexNAcHexNeuAc

Y0+HexNAc2Hex2

Y0+HexNAc3Hex2

*

*: Y0+HexNAc2Hex2NeuAc

z3 z4 z5

Y3

(2+) B6

Y5

(2+) 1338.562 (2+)

Y7

-NeuAc 1375.576 (2+)

Pr(2+) 1521.121 (2+)

-Ac 1500.107 (2+)

B4

Y3

B6

Y5

B4

D:\PA\...\180420_04 04/20/18 16:32:05 180327_01_Shoulder

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95

Time (min)


100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000

m/z

0

10

20

30

40

50

60

70

80

90

100

Re

lative

Ab

un

da

nce

204.086

274.092

366.139925.509

138.055

292.102796.866

1128.586314.0841290.6371086.476

657.234388.120 942.414 1254.587 1493.715476.134

1156.482724.354 893.348 1581.727550.174 1331.662 1655.7461427.659 1858.832

1035.459


200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000

m/z

0

10

20

30

40

50

60

70

80

90

100

Re

lative

Ab

un

da

nce

1631.659

1087.4541609.6541485.113

1302.547657.234

292.102366.141

1156.9971587.641

204.086 990.078791.377 1404.0841339.061

1678.587 1887.7151537.6041468.111

942.407468.236 1258.044614.060

B2

B3|B3 B1|B1|B1

z4 Y1

pr(3+)

1156.494(2+)

Y1,3|Y3,1 |Y2,1|Y2,1

Y1|Y1 1302.052(2+)

Y3,3 |Y2,3|Y2,3

(2+)

*

*: 1403.592(2+) -> Y1|Y 2|Y2

: 1537.093 -> z72+

●: 1561.138 -> c82+ 1484.615(2+)

Y2|Y 3|Y3 -Ac(2+)

pr(2+)

●

1586.631

z82+

Y0

Y1

Y0+HexNAcHex

Y0+HexNAc2

Y0+HexNAc2Hex

Y0+HexNAcHexNeuAc

Y0+HexNAc2Hex2

Y0+HexNAc3Hex2 B3|B3

683.413 -> b7 484.249 -> y4

770.439 -> b8 585.293 -> y5

B2 Y1

Y2

Y1

B1

B1 B1

Y1 Y1 B3 B3

Y2 Y2

454.153 B2

B2 B2

Y3 Y3


It’s a mixture.

B2|B2|B2

F:\Adam\...\180417_13 04/18/18 07:16:55 180327_04_GlcNAc

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95

Time (min)


100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000

m/z

0

10

20

30

40

50

60

70

80

90

100

Re

lative

Ab

un

da

nce

204.087

274.093

366.141

138.055

186.077

292.104

925.513

1062.156569.221 1008.142657.235 1128.595 1290.651407.168 495.182 715.280 807.380 1581.730350.147 1494.726954.459584.356 1410.666858.752


200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000

m/z

0

10

20

30

40

50

60

70

80

90

100

Re

lative

Ab

un

da

nce

1660.186

1639.186

1106.476

1514.641

657.237

204.087 366.142 1378.263 1475.630292.1041302.559715.284 1579.7041460.947

468.236

409.322

569.226 791.377 1887.7691010.744 1230.563

1058.438

961.949301.566

619.376

pr(3+)

B3

B3 Y1 z4

B1|B1

*

*: 698.268 -> HexNAc2NeuAc oxonium ion (NeuAc migration)

**: 1557.673(2+) -> Y3

Y1 1302.053(2+)

1331.081(2+)

Y1 1513.645(2+)

Y2|Y3

**

1377.915(3+)

1474.947(3+) 1579.207(2+)

-Ac(2+)

pr(2+) 1659.177(2+)

Y0 Y1

Y0+HexNAcHex

Y0+HexNAcHexNeuAc

1493.701

Y0+HexNAc2Hex

B3 B3

Y1 Y1

B3 B3

Y1

B3-Fuc Y2

Y3

B1

B1


It’s a mixture.

Y3

S-23 Additional potential structures for the HexNAc5Hex4FucNeuAc2 (2391.8569) composition: Fragments in red are missing from the EThcD spectrum

512 512

Y1

791 (2+)

B2 1736 Y1

791 (2+)

B2 1736 Y1

718.5 (2+)

B2 1881 Y1

718.5 (2+)

B2 1881

Y1

892.5 (2+) Y1

892.5 (2+)

B2 1533

B2 1533

860

860

D:\PA\...\180417_13 04/18/18 07:16:55 180327_04_GlcNAc

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95

Time (min)


100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000

m/z

0

10

20

30

40

50

60

70

80

90

100

Re

lative

Ab

un

da

nce

366.141204.087

274.093

1111.471

168.066

292.103

1289.591

924.459 1127.536657.236 1654.7171492.675569.220 731.271 1069.857948.139 1580.701528.192 1946.7961421.691407.167 1330.610880.448 1858.8051160.083316.102


200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000

m/z

0

10

20

30

40

50

60

70

80

90

100

Re

lative

Ab

un

da

nce

1111.471

1646.144

1521.607

1339.040973.919657.237

403.224 1155.9761415.9511022.365545.291 1580.647366.140292.102 790.841474.257 731.268

1481.620

219.074

S-24 1111.4371 (3+) → DFT(2407.8515)AAFPR3+ Figure S-14

Y0 Y1

Y0+HexNAcHex

Y0+HexNAc2

Y0+HexNAc2Hex

Y0+HexNAcHexNeuAc

Y0+HexNAc2Hex2

Y0+HexNAc3Hex2

1857.825 *

*:1945.815 → Y0+HexNAc2Hex2NeuAc

z3 z4 z5

Y1

(2+) B4

Y1 973.428(2+)

B5

Y5

(2+)

Y7 1338.541(2+)

B7 1387.501

-NeuAc 1521.108 (2+)

-Ac 1645.644 (2+)

Pr(2+) 1666.653

Pr(3+)

Y1

Y3

B7

Y5

B5

Y7

B3

B3

B3

F:\Adam\...\180417_15 04/18/18 11:09:17 180327_04_Shoulder

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95

Time (min)


200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000

m/z

0

10

20

30

40

50

60

70

80

90

100

Re

lative

Ab

un

da

nce

765.374

1128.005

1002.963

1532.668

657.238

204.087 606.571

892.918 1075.985

1829.796

468.234274.092

974.434

1516.610

366.141

685.796

1942.870

355.150

747.370

259.178

1623.7761403.596849.407552.609408.632

803.289

1290.600

500.859

x10 x5


200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000

m/z

0

10

20

30

40

50

60

70

80

90

100

Re

lative

Ab

un

da

nce

765.396

1531.747

1149.012

1127.999

1514.705

1002.963

715.274204.088

1342.6491241.619

1075.470

1829.7941565.729

973.949

468.238

857.395366.140

1472.585

606.576

1942.880292.104 692.792

791.379228.098

407.506 528.193

x10 x5

Y3-Fuc

B3

z4

z4

Y1

*

*: 820.398(2+) -> Y1 : 921.942(2+) -> Y2

**: B3+Fuc (Fucose migration)

***: 973.950(2+) -> Y2 -Fuc (11ppm)

●: 1055.444 -> z72+

● ●: 1104.985 -> z82+

Y1

(2+)

929.933(2+)

B1

Y3 (2+)

1046.969(2+) Y2

** *** ●

● ●

1075.483(2+) -Fuc

1127.510(2+) -Ac

1148.520(2+)

pr

Coel. (+)

Coel. (+) c5 c6

z3

292.104 B3

c3

B4

B1 z2

*

Y2 (2+)

-HexNAc -Fuc (11ppm) (2+)

-Fuc (2+)

*

*: 1046.973 -> Y3

**: 921.935(2+) -> Y1

892.939(2+)

1127.509(2+) -Ac

1148.512(2+) pr

c5 c6

B3 Y1

Y2

Y1

Y3

B4

B3 Y1

Y2

Y3

**

Y1

Y2


Y2

Y2

S-26 Additional potential structures for the HexNAc3Hex2FucNeuAc (1370.4971) composition: Fragments in red are missing from the EThcD spectrum

Y1

892.5 (2+)

B2 512 Y1

718.5 (2+)

B3 860

Neutral loss of HexNeuAc (921 (2+)) from the

precursor is present which cannot be explained with

this structure.

Documents

Novel O-linked Sialoglycan Structures in Human Urinary ... · S-1 SUPPORTING INFORMATION for the article “Novel O-linked Sialoglycan Structures in Human Urinary Glycoproteins”