N

O ONH

O

O

-ON

O

NH

HNO

O

O-

Fe3+

A B C D

R2

R1

N+

OH

EF

N

O ONH

O

O

HN

O

NH

O-NO

OR1

Fe3+E

e-R2

B CA

DO-

N+

H

F

proximal ferri-monodeoxymycobactindistal ferri-monodeoxymycobactin

Madigan et al. Fig. S2A

calculated observed R1 = C19H37 calculated observedfragment m/z m/z R2 = CH3 fragment m/z m/z

A 650.3620 650.3602 A 665.3491 665.3487B 678.3575 678.3549 B 693.3441 693.3415C 696.3675 696.3665 C 711.3546 711.3540D 376.3579 376.3570 D 155.0815 155.0802E 504.1307 504.1294 E 355.0620 355.0592F 84.0808 84.0817 F 100.0757 100.0756

calculated observed R1 = C19H37 calculated observedfragment m/z m/z R2 = H fragment m/z m/z

A 636.3458 636.3444 A 651.3329 651.3323B 664.3407 664.3431 B 679.3278 679.3274C 682.3513 682.3503 C 697.3384 697.3380D 376.3579 376.3572 D 155.0815 155.0811E 490.1145 490.1143 E 341.0457 341.0458F 84.0808 84.0810 F 100.0757 100.0750

calculated observed R1 = C17H33 calculated observedfragment m/z m/z R2 = CH3 fragment m/z m/z

A 622.3302 622.3289 A 637.3173 637.3162B 650.3251 650.3265 B 665.3122 665.3066C 668.3357 668.3338 C 683.3227 683.3162D 348.3261 348.3269 D 155.0815 155.0845E 504.1302 504.1283 E 355.0620 355.0604F 84.0808 84.0809 F 100.0757 nd

calculated observed R1 = C17H33 calculated observedfragment m/z m/z R2 = H fragment m/z m/z

A 608.3145 608.3140 A 623.3016 623.3014B 636.3094 636.3079 B 651.2965 651.2832C 654.3200 654.3209 C 669.3071 669.3089D 348.3261 348.3249 D 155.0815 155.0814E 490.1145 490.1126 E 341.0457F 84.0808 84.0809 F 100.0757 nd

m/z 865.4305: C18:1 (serine)

m/z 879.4486: C18:1 (α-methyl serine)

m/z 893.4586: C20:1 (serine)

m/z 907.4820: C20:1 (α-methyl serine)

341.0440

nd = not detected

A

N

O ONH

O

O

-ON

O

NH

HNO

O

O-

Fe3+

651.3329679.3278

697.3384 155.0815

N+

OH

341.0457100.0757

C19H37

0

1

2

651.3323 893.4585

697.3380

341.0458155.0811

100 200 300 400 500 600 700 800 900

coun

ts x

104

m/z

679.3274

C

N

O ONH

O

O

HN

O

NH

O-NO

O

Fe3+490.1145

e-

664.3407682.3513636.3458

376.3579O-

N+

H

84.0808

Madigan et al. Fig. S2B-C

C19H37

0

1

2

893.458484.0810

682.3503

636.3444

376.3572

100 200 300 400 500 600 700 800 900

ferri-monodeoxymycobactin C20:1 (serine), distal isomer

coun

ts x

104

m/z

664.3431

490.1143

B

0

1

2100.0750

99.5 100 100.5m/z

coun

ts x

102

100.0750

ferri-monodeoxymycobactin C20:1 (serine), proximal isomer

N

O ONH

O

O

-ON

O

NH

HNO

O

O-

Fe3+

637.3173 665.3122 683.3227 155.0815

355.0620

C17H33

0

1

2

3

4

5

637.3157

683.3162 879.4434

355.0604

100 200 300 400 500 600 700 800 900

ferri-monodeoxymycobactin C18:1 (α-methyl serine), proximal isomer

coun

ts x

102

m/z

155.0845

665.3066

E

N

O ONH

O

O

HN

O

NH

O-NO

O

Fe3+504.1302

e-

650.3251668.3357622.3302

348.3261O-

N+

H

84.0808

Madigan et al. Fig. S2D-E

C17H33

0

2

4

6

8

879.442484.0809

668.3338

348.3269504.1283

100 200 300 400 500 600 700 800 900

ferri-monodeoxymycobactin C18:1 (α-methyl serine), distal isomer

coun

ts x

103

m/z

650.3265

622.3289

D

N

O ONH

O

O

-ON

O

NH

HNO

O

O-

Fe3+

623.3016651.2965 669.3071 155.0815

341.0457

N

O ONH

O

O

HN

O

NH

O-NO

O

Fe3+490.1145

e-

636.3094 654.3200608.3145

348.3261O-

N+

H

84.0808

proximal isomerdistal isomer

Madigan et al. Fig. S2F

C17H33 C17H33

0

1

2

3

ferri-monodeoxymycobactin, C18:1 (serine), proximal and distal isomers865.4281

84.0809

654.3209

348.3249

490.1126155.0814

623.3014

100 200 300 400 500 600 700 800

coun

ts x

103

608.3140

669.3089341.0440

m/z

636.3079651.2832

F

N

O ONH

O

O

HN

O

NH

OHNO

OR1

OH H+

CA

R2

B

N+

H

E

D N

O ONH

O

O

HON

O

NH

HNO

O

OHH+

A B C

R2

R1

N+

OH

E

D

proximal apo-monodeoxymycobactindistal apo-monodeoxymycobactin

calculated observed R1 = C19H37 calculated observedfragment m/z m/z R2 = CH3 fragment m/z m/z

A 596.4422 596.4588 A 612.4371 612.4347B 624.4371 624.4445 B 640.4320 640.4385C 642.4477 642.4579 C 658.4426 658.4441D 376.3574 376.3623 D 392.3523 392.3560E 84.0808 84.0828 E 100.0757 100.0778

calculated observed R1 = C19H39 calculated observedfragment m/z m/z R2 = CH3 fragment m/z m/z

A 598.4578 598.4674 A 614.4527

nd

B 626.4527 626.4588 B 642.4477 642.4516C 644.4633 644.4699 C 660.4582 660.4564D 378.3730 378.3774 D 394.3680E 84.0808 84.0828 E 100.0757 100.0771

calculated observed R1 = C19H39 calculated observedfragment m/z m/z R2 = H fragment m/z m/z

A 584.4422 584.4206 A 600.4371 ndB 612.4371 612.4349 B 628.4320 628.4394C 630.4477 630.4539 C 646.4426 646.4446D 378.3730 378.3787 D 394.3680 ndE 84.0808 84.0827 E 100.0757 100.0769

m/z 842.5613: C20:0 (serine)

m/z 856.5775: C20:0 (α-methyl serine)

m/z 854.5663: C20:1 (α-methyl serine)

Madigan et al. Fig. S2G

614.4320

calculated observed R1 = C H calculated observedfragment m/z m/z R2 fragment m/z m/z

A AB BC CD DE 84.0808 84.0828 E 100.0757 100.0747

m/z 812.5161: C18:1 (serine)

554.3952582.3901600.4007348.3261

554.3844582.3842600.4072348.3239

= H17 33

570.3901598.3851616.3956364.3210

598.3832616.3940364.3227

nd

nd = not detected

G

N

O ONH

O

O

HN

O

NH

OHNO

O

OH H+

642.4477596.4422624.4371

N+

H

84.0808

376.3574

Madigan et al. Fig. S2H-I

C19H37

0

1

2

854.5736376.3623

624.444584.0828

100 200 300 400 500 600 700 800

coun

ts x

103

642.4579

596.4588

apo-monodeoxymycobactin C20:1 (α-methyl serine), distal isomer

m/z

H

N

O ONH

O

O

HON

O

NH

HNO

O

OHH+

612.4371640.4320

658.4426

N+

OH

100.0705

392.3523

C19H37

0

0.5

1

1.5

854.5735640.4385

392.3560

100.0778

100 200 300 400 500 600 700 800

coun

ts x

103

658.4441612.4347

apo-monodeoxymycobactin C20:1 (α-methyl serine), proximal isomer

m/z

I

N

O ONH

O

O

HN

O

NH

OHNO

O

OH H+

644.4633598.4578626.4527

N+

H

84.0808

378.3730

Madigan et al. Fig. S2J-K

C19H39

0

2

4

684.0828

378.3774644.4699

100 200 300 400 500 600 700 800

coun

ts x

102

m/z

apo-monodeoxymycobactin C20:0 (α-methyl serine), distal isomer

856.5855

598.4674

626.4588

J

N

O ONH

O

O

HON

O

NH

HNO

O

OHH+

614.4527642.4477

660.4582

N+

OH

100.0757

C19H39

0

1

2

3

4

642.4516100.0771 614.4320

660.4564

100 200 300 400 500 600 700 800

coun

ts x

102

856.5843apo-monodeoxymycobactin C20:0 (α-methyl serine), proximal isomer

m/z

K

N

O ONH

O

O

HN

O

NH

OHNO

O

OH H+

630.4477584.4422612.4371

N+

H

84.0808

378.3730

Madigan et al. Fig S2L-M

0

1

2

842.547584.0827

378.3787 630.4483584.4206

100 200 300 400 500 600 700 800

coun

ts x

102

m/z

612.4349

apo-monodeoxymycobactin C20:0 (serine), distal isomer

C19H39

L

N

O ONH

O

O

HON

O

NH

HNO

O

OHH+

628.4320 646.4426

N+

OH

100.0757

0

0.5

1

842.5719

100.0769

628.4394646.4446

100 200 300 400 500 600 700 800

apo-monodeoxymycobactin C20:0 (serine), proximal isomer

coun

ts x

102

m/z

C19H39

M

N

O ONH

O

O

HN

O

NH

OHNO

O

OH H+

600.4007554.3952582.3901

N+

H

84.0808

348.3261 N

O ONH

O

O

HON

O

NH

HNO

O

OHH+

616.3956598.3851

N+

OH

100.0757

364.3210

proximal isomerdistal isomer

Madigan et al Fig. S2N

C H17 33 C H17 33

0

2

4

6

8812.5075

348.323984.0814

600.4072

100 200 300 400 500 600 700 800

coun

ts x

102

m/z

616.3940

598.3832

364.3227100.0747

582.3842

554.3844

apo-monodeoxymycobactin C18:1 (serine), distal and proximal isomers

N

OH

N

O ONH

O

O

HN

O

NH

OHNO

OCOO

H+

BA C

N+

H

DE

N+

O

OH

N

O ONH

OH

O

H2N

H+F

distal apo-monodeoxycarboxymycobactinR1

R2

R3

R2

* *

R1 = C6H10 R2 = CH3 R3 = CH3

calculated observedfragment m/z m/z

AB

472.2442 472.2334

C500.2391 500.2351

D518.2497 518.2452350.1710 350.1673332.1605 332.1626333.1445 333.1448252.1594 252.1550220.1332 220.129884.0808 84.0774

-MeOH 698.3396 698.3336

R1 = C7H14 R2 = CH3 R3 = Hcalculated observed

fragment m/z m/z

AB

474.2599

C502.2548 502.2487

D520.2653 520.2638350.1710

D-H2O 332.1605D-NH3 333.1445

E 254.1751 254.1705F 84.0808 84.0799

m/z 730.3668: methyl ester of C8:1 (methyl)

m/z 732.3789: C9:0 (methyl)R1 = C5H8 R2 = CH3 R3 = CH3

calculated observedfragment m/z m/z

AB

458.2286

C486.2235 486.2224

D504.2340 504.2330350.1710332.1605 332.1530333.1445 333.1449238.1438 238.1419206.1176 206.116384.0808 84.0786

-MeOH 684.3239 684.3303

R1 = C5H8 R2 = H R3 = CH3

calculated observedfragment m/z m/z

AB

444.2129

C472.2078490.2184 490.2191

D 336.1554 336.1543D-H2O 318.1448 318.1326D-NH3 319.1288 319.1298

E 238.1438 238.1424E-MeOH 206.1176 206.1165

F 84.0808 84.0800-MeOH 670.3083

m/z 716.3497: methyl ester of C7:1 (methyl)

m/z 702.3304: methyl ester of C7:1 (serine)

Madigan et al. Fig. S2O

* R could also be on this carbon

2

nd

COOR1 R3

ndnd

ndnd

nd

ndnd

nd

nd = not detected

-MeOH

O

D-H2OD-NH3

EE-MeOH

F

D-H2OD-NH3

EE-MeOH

F

OH

N

O ONH

O

O

HN

O

NH

OHNO

OCOOCH

H+

N+

H

N+

O

OH

N

O ONH

OH

O

H2N

H+

C H 3

Madigan et al. Fig. S2P-Q

5 8

-MeOH

238.1438

206.1176

84.0808

332.1605

-H O -NH

333.1445

32

504.2340

486.2235

COOCHC H 35 8

**

0

1

2238.1419

84.0786504.2330

716.3563333.1449 684.3303

100 200 300 400 500 600 700

apo-monodeoxycarboxymycobactin methyl ester of C7:1 (methyl), distal isomer

coun

ts x

102

m/z

486.2224332.1530

206.1163

* methyl could be on either carbon

-MeOH = 684.3239

350.1710

P OH

N

O ONH

O

O

HN

O

NH

OHNO

OCOOCH

H+

N+

H

N+

O

OH

N

O ONH

OH

O

H2N

H+

C H 35 8

-MeOH

238.1438

206.1176

84.0808

318.1448

-H O -NH

319.1288

32

490.2184

COOCHC H 35 8

0

0.5

1

84.0800

238.1424

490.2191

319.1298 702.3621

100 200 300 400 500 600 700

coun

ts x

102

m/z

apo-monodeoxycarboxymycobactin methyl ester of C7:1 (serine), distal isomer

206.1165

336.1543

318.1326

336.1554

Q

OH

N

O ONH

O

O

HN

O

NH

OHNO

OCOOCH

H+

N+

H

N+

O

OH

N

O ONH

OH

O

H2N

H+

C H 3

Madigan et al. Fig. S2R-S

6 10

-MeOH

252.1594

220.1332

84.0808

350.1710

332.1605

-H O -NH

333.1445

32

518.2497500.2391

472.2442

COOCHC H 36 10

0

2

4

6

8 252.1550

84.0774730.3628

518.2452

333.1448

698.3336

100 200 300 400 500 600 700

coun

ts x

102

m/z

500.2351

472.2334

350.1673

332.1626

apo-monodeoxycarboxymycobactin methyl ester of C8:1 (methyl), distal isomer

220.1298

* methyl could be on either carbon

*

-MeOH = 698.3396

*

R

OH

N

O ONH

O

O

HN

O

NH

OHNO

OCOOH

H+

N+

H

N+

O

C H7 14

254.1751

84.0808

520.2653502.2548

COOHC H7 14

**

* methyl could be on either carbon

0

1

2

84.0799

254.1705

732.3763

520.2638

100 200 300 400 500 600 700

coun

ts x

102

m/z

apo-monodeoxycarboxymycobactin C9:0 (methyl), distal isomer

502.2487

S