detection, differentiation, and subtyping of botulinum ... · detection, differentiation, and...

Detection, Differentiation, and Subtyping of Botulinum Neurotoxins

with Mass Spectrometry

Suzanne R. Kalb*, Hercules Moura*, Wanda I. Santana*, Jakub Baudys*, Maria I. Solano*, Adrian R. Woolfitt*, Theresa J. Smith†, James D. Marks#, James L.

Pirkle*, Leonard A. Smith†, and John R. Barr**Centers for Disease Control and Prevention

†USAMRIID#UCSF

Botulism Types

Foodborne

Infant (& adult colonization)

Wound

Intentional contamination

Toxicity of Botulinum NeurotoxinToxicity of Botulinum Neurotoxin

AGENTLD50

(mg/kg) SOURCEBotulinum

Toxin0.001 Bacterium

Tetanus Toxin

0.002 Bacterium

Ricin 3 Plant (Castor bean)

VX 15 Chemical Agent

Soman (GD) 64 Chemical Agent

Sarin (GB) 100 Chemical Agent

T-2 Toxin 1210 Fungal Mycotoxin

TherapeuticsTherapeutics

& Aesthetics& Aesthetics

Traditional Diagnosis of Botulism

•Preparation of extract

•IP injection into 2 mice

•Unneutralized

•Neutralized with

specific antitoxin

•4 day monitor of mice

for symptoms

SNARE Complex Function

Ca++Ca++

Ca++

Syntaxin 1ABoNT-C

BoNT-B BoNT-D

BoNT-F

Synaptobrevin

Synaptic Vesicle

BoNT-A

BoNT-C

BoNT-E

SNAP-25

ACh

ACh

AChACh

ACh

• SNARE complex forms between synaptic vesicle and neuronal membrane• Synaptic Vesicle carrying neurotransmitter fuses with neuronal membrane• Nerve impulse is transmitted

BoNT-G

Endopep-MS Method

BoNT A

A and C Substrate

B and G Substrate

E Substrate

D and F Substrate

Specificity of Toxin Cleavage

Biotin-KGSNRTRIDEANQRATRMLGGK-BiotinBiotin-KGSNRTRIDEANQ

RATRMLGGK-Biotin

IIGNLRHMALDMGNEIDTQNRQIDRIMEKADIIGNLRHMALDMGNEIDTQNRQIDR

IMEKAD

LSELDDRADALQAGASQFETSAAKLKRKYWWKNLKLSELDDRADALQAGASQ

FETSAAKLKRKYWWKNLK

AQVDEVVDIMRVNVDKVLERDQKLSELDDRADALQAGASAQVDEVVDIMRVNVDKVLERDQ

KLSELDDRADALQAGAS

2912.61713.81215.3

3612.12924.3705.8

4039.61759.82297.7

4312.82570.91759.9

BoNT-ASubstrate

BoNT-ESubstrate

BoNT-BSubstrate

BoNT-FSubstrate

BoNT-A Mass

BoNT-F

BoNT-B

BoNT-E

Shortened Targets for BoNT

Endopep-MS Reaction Conditions

•Reaction buffer is 50 mM Hepes (pH 7.3), 25 mM DTT, 20 μM ZnCl2 , and 1 mg/mL BSA•Reaction volume is 20 μL•After incubation, reaction is diluted 1:10 in matrix solution, 5 mg/mL HCCA

Detection of BoNT Activity

•Enzymatic reaction mixed with matrix compound•Mixture spotted onto stainless steel MALDI plate and allowed to dry•Plate analyzed by MALDI-TOF/MS on Applied Biosystems 4800 Proteomics Analyzer•Analysis is 15 seconds per sample



m/z

IntAbsence of BoNT



m/z

IntBoNT present

Mouse Or Machine?Mouse Or Machine?No

Toxin No No

ToxinToxin

Toxin Present Toxin Toxin

PresentPresent

m/zm/zm/z

m/zm/zm/z

No Toxin No No

ToxinToxin

Toxin Present Toxin Toxin

PresentPresent

m/z

IntAbsence of BoNT

m/z

IntBoNT present

m/z

Int Serum or stool sample

Detection of BoNT Activity by MS

Antibodies for Botulinum Neurotoxin A, B, E, F were prepared at the

University or California at San Francisco By Prof. Jim Marks from his yeast library

The antibodies are now recombinant, have high affinities, and are selective

for the BoNT serotypes

Begin with blocked Ab- coated beads

Incubate BoNT in matrix with beads

Wash beads to remove matrix

Incubate beads with buffer and substrate Analyze

supernatant by mass spectrometry

Antibody Extraction of BoNT

BoNT A and B

1100 1520 1940 2360 2780 3200Mass (m/z)

1.9E+4

0102030405060708090

100

% In

tens

ity

1197.73

1699.88

2878.56

BoNT A

BoNT B

1100 1780 2460 3140 3820 4500Mass (m/z)

9.6E+3

0102030405060708090

100

% In

tens

ity

1760.06

2283.57

1742.041142.76 1798.01 4024.762305.56 2825.91 3169.07 3564.36

BoNT E and F

1100 1880 2660 3440 4220 5000Mass (m/z)

1768.5

0102030405060708090

100

% In

tens

ity

2923.44

1136.57 4041.01

BoNT E

BoNT F

1100 1880 2660 3440 4220 5000Mass (m/z)

7058.5

0102030405060708090

100

% In

tens

ity

1345.65

3168.521327.64

2249.06 4495.261412.72 3151.521872.86 2641.28

Workflow of Endopep-MS Assay

Sample & Reaction Prep 30 min

Sample Size = 8 or less

BoNT extraction 1 hr

Endopep-MS Reaction 4 hrs

Total time = 5.75 hrs

Analysis of Endopep-MS Reaction 15 min

Sample Size = 8 to 96

Sample & Reaction Prep 1 hr



Total time = 6.75 hrs

Analysis of Endopep-MS Reaction 45 min

Botulinum Neurotoxin Tree

Botulinum Neurotoxin (BoNT)

BoNT A BoNT B BoNT E BoNT FBoNT C BoNT D BoNT G 30% toxin type

A1 A2 A3 A4 84% subtype

BoNT A

Detection of Subtypes

BoNT A•A1•A2•A3•A4

BoNT B•B1•B2•bivalent B•Np B

BoNT E•E1•E2•E3•E It butyricum•E Ch butyricum

BoNT F•prot F•np F•bivalent F•F baratii

BoNT C•C1•Mosaic C

BoNT D•D1•Mosaic D

BoNT G•G1

LOD in serum—BoNT A and B

1100 1520 1940 2360 2780 3200Mass (m/z)

4189.8

0102030405060708090

100

% In

tens

ity 2878.59

2254.291595.02

1853.101197.801190 1194 1198 1202 1206 1210

Mass (m/z)

123.6

0

50

100

% In

tens

ity

1197.80

0.25U BoNT A in 500 uL serum

1100 1840 2580 3320 4060 4800Mass (m/z)

1554.7

0102030405060708090

100

% In

tens

ity 2013.14

4023.28

1759.503167.97

1750 1754 1758 1762 1766 1770Mass (m/z)

450.8

0

50

100

% In

tens

ity

1759.50

0.05U BoNT B in 500 uL serum

LOD in serum—BoNT E and F

1100 1840 2580 3320 4060 4800Mass (m/z)

1076.9

0102030405060708090

100

% In

tens

ity

1252.63

1136.51

4040.802140.94 2923.30

2915 2919 2923 2927 2931 2935Mass (m/z)

302.5

0

50

100

% In

tens

ity 2923.30

1100 1840 2580 3320 4060 4800Mass (m/z)

1814.5

0102030405060708090

100

% In

tens

ity

2249.07

1345.652239.58

1335 1340 1345 1350 1355 1360Mass (m/z)

255.3

0

50

100

% In

tens

ity

1345.65

0.05U BoNT F in 500 uL serum

0.05U BoNT E in 500 uL serum

LOD in stool extract--BoNT A and B

1100 1520 1940 2360 2780 3200Mass (m/z)

8789.4

0102030405060708090

100

% In

tens

ity 2878.53

2254.201594.912410.29 2860.531440.27 2098.101852.991197.70

1100 1840 2580 3320 4060 4800Mass (m/z)

2686.1

0102030405060708090

100

% In

tens

ity

1833.13

4024.20

1759.88

2U BoNT A in 500 uL stool extract

1U BoNT B in 500 uL stool extract

LOD in stool extract—BoNT E and F

1100 1840 2580 3320 4060 4800Mass (m/z)

3325

0102030405060708090

100

% In

tens

ity

4041.04

2022.001137.66

3610.80 4023.062922.36

5U BoNT E in 500 uL stool extract

0.25U BoNT F in 500 uL stool extract

1100 1840 2580 3320 4060 4800Mass (m/z)

926.6

0102030405060708090

100

% In

tens

ity

2249.05

1873.84

2641.272240.041345.63 2085.97 2542.19 3040.43

Real World Samples—BoNT A

1100 1520 1940 2360 2780 3200

Mass (m/z)

6766.0

0102030405060708090

100

% In

tens

ity

1197.83

1700.02

1267.82

Carrot juice

1100 1520 1940 2360 2780 3200

Mass (m/z)

2077.8

0102030405060708090

100

% In

tens

ity

1197.77

1699.95

Serum from carrot juice patient

More Real World Samples

1100 1840 2580 3320 4060 4800Mass (m/z)

2208.6

0102030405060708090

100

% In

tens

ity

1554.762923.44

1137.612021.48 4041.23

BoNT E in serum

1100 1840 2580 3320 4060 4800Mass (m/z)

3910.1

0102030405060708090

100

% In

tens

ity

1759.82

2283.24

4024.04

BoNT B in stool





BoNT A

Tryptic Digestion

KR

R

K

R

K

RK

R

R

K

K

R

K

K

R

R

R

R

R

R

K

K

K

K

K

R

Fragmentation Nomenclature

H2N CH

C

O

NH

CH

C

O

NH

CH

C

O

NH

CH

C

O

OH

R1 R2 R3 R4

b1

y3

b2

y2

b3

y1

Biemann, K., Biomed. Mass Spectrom. 16 (1988) 99; Biemann, K. in Methods in EnzymologyMass Spectrometry, McCloskey, J.A. Ed.; Academic Press, San Diego (1990) pp. 886.887.

Sequence of Subtype A1PFVNKQFNYKDPVNGVDIAYIKIPNVGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNP PPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGGS TIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGYG STQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPNR VFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKAK SIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKVL NRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFTG LFEFYKLLCVRGIITSKTKSLDKGYNKALNDLCIKVNNWDLFFSPSEDNFTNDLNKGEEI TSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNGK KYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEAA MFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGALIFSGA VILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAKV NTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDLSSKLNESINKAM ININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDKV NNTLSTDIPFQLSKYVDNQRLLSTFTEYIKNIINTSILNLRYESNHLIDLSRYASKINIG SKVNFDPIDKNQIQLFNLESSKIEVILKNAIVYNSMYENFSTSFWIRIPKYFNSISLNNE YTIINCMENNSGWKVSLNYGEIIWTLQDTQEIKQRVVFKYSQMINISDYINRWIFVTITN NRLNNSKIYINGRLIDQKPISNLGNIHASNNIMFKLDGCRDTHRYIWIKYFNLFDKELNE KEIKDLYDNQSNSGILKDFWGDYLQYDKPYYMLNLYDPNKYVDVNNVGIRGYMYLKGPRG SVMTTNIYLNSSLYRGTKFIIKKYASGNKDNIVRNNDRVYINVVVKNKEYRLATNASQAG VEKILSALEIPDVGNLSQVVVMKSKNDQGITNKCKMNLQDNNGNDIGFFIGFHQFNNIAKLVASNWYNRQIERSSRTLGCSWEFIPVDDGWGERPL

PFVNKQFNYKDPVNGVDIAYIKIPNVGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNP PPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGGS TIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGYG STQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPNR VFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKAK SIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKVL NRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFTG LFEFYKLLCVRGIITSKTKSLDKGYNKALNDLCIKVNNWDLFFSPSEDNFTNDLNKGEEI TSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNGK KYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEAA MFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGALIFSGA VILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAKV NTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDLSSKLNESINKAM ININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDKV NNTLSTDIPFQLSKYVDNQRLLSTFTEYIKNIINTSILNLRYESNHLIDLSRYASKINIG SKVNFDPIDKNQIQLFNLESSKIEVILKNAIVYNSMYENFSTSFWIRIPKYFNSISLNNE YTIINCMENNSGWKVSLNYGEIIWTLQDTQEIKQRVVFKYSQMINISDYINRWIFVTITN NRLNNSKIYINGRLIDQKPISNLGNIHASNNIMFKLDGCRDTHRYIWIKYFNLFDKELNE KEIKDLYDNQSNSGILKDFWGDYLQYDKPYYMLNLYDPNKYVDVNNVGIRGYMYLKGPRG SVMTTNIYLNSSLYRGTKFIIKKYASGNKDNIVRNNDRVYINVVVKNKEYRLATNASQAG VEKILSALEIPDVGNLSQVVVMKSKNDQGITNKCKMNLQDNNGNDIGFFIGFHQFNNIAKLVASNWYNRQIERSSRTLGCSWEFIPVDDGWGERPL

Sequence of Subtype A1

Tryptic Peptide from A1 and A2

SFGHDVLNLTRNL: 2.79E5

200 300 400 500 600 700 800 900 1000 1100 1200m/z

0

25

50

75

100

Rel

ativ

e A

bund

ance

512.851 830.355

621.036

484.170

715.297

544.092643.214 1024.439429.037 756.180276.215 983.262 1084.505870.312

y7

y6b5

b4

SFGHEVLNLTRNL: 1.05E3

200 300 400 500 600 700 800 900 1000 1100 1200m/z

0

25

50

75

100

Rel

ativ

e A

bund

ance

629.421

492.162

844.643

715.301

558.303429.041 997.604770.464 1038.766276.213 884.525

y7

y6b4 b5

Subtype Specific Peptides

m/z of A1 sequence m/z of A2 sequence

958.3 GTLIGQVDR 823.3 RYIMIK

1272.3 SFGHEVLNLTR 1050.3 LKDFDASVR

1346.3 YESNHLIDLSR 1258.3 SFGHDVLNLTR

1355.3 VYTFFSSDYVK 1328.4 VNTQIDLIREK

1474.4 VNYTIYDGFNLR 1485.4 VYYDSIDKNQIY

1711.5 IALTNSVNEALLNPSR 1529.4 IPNAGQMQPVKAFK

1733.5 RLEDFDASLKDALLK 1937.4 GANLSTNFNGQNTEINSR

1818.4 GSVMTTNIYLNSSLYR 2127.6 FATDPAVTLAHELIHAEHR

2055.6 FATDPAVTLAHELIHAGHR 2153.6 INIGDRVYYDSIDKNQIK

BoNT B Detected in Two Patients

Louisiana Case

1100 1780 2460 3140 3820 4500Mass (m/z)

1438.6

0102030405060708090

100

% In

tens

ity

2283.32

2825.571759.89

2498.41 4024.18

Mass (m/z)

Hawaii Case

1100 1780 2460 3140 3820 4500

4387.9

0102030405060708090

100

% In

tens

ity

1759.79

2283.18

1741.771217.57 1781.77 2825.382299.16 4024.86

MS/MS of Peptides

NL: 1.97E4

400 600 800 1000 1200 1400 1600 1800 2000m/z0

50

100

Rel

ativ

e Ab

unda

nce

y8

y9

y10 y11 y12y13 y14

y7

y6

y5y3

Louisiana Case = B1YFSIFNTELSQSNIEER

y10NL: 2.76E2

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

0

50

100

Rel

ativ

e Ab

unda

nce

y11 y12

y13

y14 y15y9

y8

y5

Hawaii Case = bvBYFSIFNTELSQSNIEEIYK





BoNT A

A1

99.7% strainMany strains with very similar sequences

Sequence of A1 Hall StrainPFVNKQFNYKDPVNGVDIAYIKIPNVGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNP PPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGGS TIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGYG STQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPNR VFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKAK SIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKVL NRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFTG LFEFYKLLCVRGIITSKTKSLDKGYNKALNDLCIKVNNWDLFFSPSEDNFTNDLNKGEEI TSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNGK KYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEAA MFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGALIFSGA VILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAKV NTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDLSSKLNESINKAM ININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDKV NNTLSTDIPFQLSKYVDNQRLLSTFTEYIKNIINTSILNLRYESNHLIDLSRYASKINIG SKVNFDPIDKNQIQLFNLESSKIEVILKNAIVYNSMYENFSTSFWIRIPKYFNSISLNNE YTIINCMENNSGWKVSLNYGEIIWTLQDTQEIKQRVVFKYSQMINISDYINRWIFVTITN NRLNNSKIYINGRLIDQKPISNLGNIHASNNIMFKLDGCRDTHRYIWIKYFNLFDKELNE KEIKDLYDNQSNSGILKDFWGDYLQYDKPYYMLNLYDPNKYVDVNNVGIRGYMYLKGPRG SVMTTNIYLNSSLYRGTKFIIKKYASGNKDNIVRNNDRVYINVVVKNKEYRLATNASQAG VEKILSALEIPDVGNLSQVVVMKSKNDQGITNKCKMNLQDNNGNDIGFFIGFHQFNNIAKLVASNWYNRQIERSSRTLGCSWEFIPVDDGWGERPL

Tryptic Peptide from A1 Hall and A1(B)

IPNAGQMQPVKNL: 5.55E5

200 300 400 500 600 700 800 900 1000 1100 1200m/z0

25

50

75

100

Rel

ativ

e Ab

unda

nce

549.27

787.33

1000.45343.30

886.34602.35540.67 868.35 1064.33424.20212.21 730.47934.30

b3b4

y7

y8

IPNVGQMQPVKNL: 7.88E5

200 300 400 500 600 700 800 900 1000 1100m/z0

25

50

75

100

Rel

ativ

e Ab

unda

nce

535.51

787.37526.78 972.46343.22 858.41

1036.55

602.45

212.19 396.34

y7 y8b3b4

Workflow of Endopep-MS Assay

Sample & Reaction Prep 30 min

Sample Size = 8 or less



Diagnosis of botulism and serotype at 5.75 hrsAnalysis of Endopep-MS Reaction 15 min

Digest of toxin on beads 10 min

Analysis of digest of toxin 1.5 hrsIdentification of subtype at 7.5 hrs

Shortened Assay—30 minutes

• BoNT A at a concentration estimated to be the human LD50 in 8 oz of milk was spiked into milk

• Activity assay from start to finish was performed in 30 minutes

• Spectrum shows clear evidence for BoNT A

1100 1520 1940 2360 2780 3200Mass (m/z)

534.4

0102030405060708090

100

% In

tens

ity

2878.38

1440.191197.65

1699.76

AA

Shortened Assay

• BoNT A1 and A2 were spiked into milk• Activity assay from start to finish was

performed in 30 minutes• Spectra show clear evidence for BoNT A• Tryptic digestion and analysis were

performed in an additional 30 minutes• Within one hour, two samples were

identified as positive for BoNT A and were differentiated as BoNT A1 or A2

1000 1440 1880 2320 2760 3200Mass (m/z)

4781.0

0102030405060708090

100

% In

tens

ity

1197.69

1699.85

2878.56

1000 1440 1880 2320 2760 3200Mass (m/z)

2551.6

0102030405060708090

100

% In

tens

ity 2878.56

1197.69

1699.83

A1

A2

30 Minute Assay Detects BoNT A

Mass (m/z)990 1232 1474 1716 1958 2200

2.0E+4

0102030405060708090

100

% In

tens

ity

1087.59

1312.621140.76

1434.781158.591029.61

1397.701071.63 1907.00 2056.151711.981193.65 1892.051600.87 1733.991474.77

1272.70

990 1232 1474 1716 1958 2200Mass (m/z)

3.2E+4

0102030405060708090

100

% In

tens

ity

1087.57

1140.741312.60

1434.761029.59 1158.57 1397.69 1906.97

1600.852128.131258.67

1000.58

A1

A2

1 Hour Assay Differentiates BoNT A

Different MS Options

MALDI-TOF MS

LC-MS/MS

Faster analysis (15 sec)

Can multiplex and quantitate

Detection of BoNT A in Milk by LC- MS/MS

1 1.2 1.4 1.6 1.8 2 2.2 2.4 2.6 Time, minBlank

0.5 U1 U

5 U10 U

Rel

ativ

e In

tens

ity

NT product peptide CT product peptide

Detection of BoNT B in Serum by LC-MS/MS

1.7 1.8 1.9 2 2.1 2.2Blank

0.1 U0.5 U

1 U5 U

Time, min

Rel

ativ

e In

tens

ity

NT product peptide

Detection of BoNT E in Serum by LC-MS/MS

1.8 2 2.2 2.4 2.6 2.8Blank

10 U50

1005001000

5000 U

Time, min

Rel

ativ

e In

tens

ity

BoNT E NT product, high-m/z MRM

Detection of BoNT F in Stool by LC-MS/MS

1.2 1.6 2 2.4 2.8 3.2 3.6Blank

0.5 U1 U

5 U10 U

Time, min

CT product peptide

NT product peptide

Rel

ativ

e In

tens

ity

4E17.1 Monoclonal Antibody

• Human monoclonal which cross-reacts with BoNT/A, /B, /E, and /F

• Selected from an immune human antibody library from a donor immunized with pentavalent toxoid

• Binds an epitope on the translocation domain

Detection of Subtypes with 4E17.1 Ab

BoNT A•A1•A2•A3•A4

BoNT B•B1•B2•bivalent B•Np B

BoNT E•E1•E2•E3•E It butyricum•E Ch butyricum

BoNT F•prot F•np F•bivalent F•F baratii

BoNT C•C1•Mosaic C

BoNT D•D1•Mosaic D

BoNT G•G1

Limits of Detection

1190 1194 1198 1202 1206 1210Mass (m/z)

29.5

0

100

% In

tens

ity

1197.400.5U A1

1750 1755 1760 1765 1770 1775Mass (m/z)

54.5

0

100

% In

tens

ity

1760.01 0.05U B1

2910 2917 2924 2931 2938 2945Mass (m/z)

107.0

0

100

% In

tens

ity

2922.57

0.1U E3

1335 1340 1345 1350 1355 1360Mass (m/z)

48.7

0

100

% In

tens

ity

1345.69

1342.75

0.05U protF

Multiplexed Detection of BoNT ABEF

1100 1540 1980 2420 2860 3300Mass (m/z)

378.2

0102030405060708090

100

% In

tens

ity

EE

AA B

B

F

F

BoNT ABEF spiked into serum

BoNT B spiked into milk

1100 1540 1980 2420 2860 3300Mass (m/z)

59.5

0102030405060708090

100

% In

tens

ity

1759.84

2283.26

B

B

Detection of BoNT bivalent strains

1100 1360 1620 1880 2140 2400Mass (m/z)

664.2

0102030405060708090

100

% In

tens

ity

1197.87

1760.04

1700.01

2283.62

A

A

B

B

A2b

1100 1360 1620 1880 2140 2400Mass (m/z)

1002.3

0102030405060708090

100

% In

tens

ity

1760.01

2283.65

1197.851700.03

A A

B

B

Ba4

Detection of BoNT bivalent strains

Af

1100 1360 1620 1880 2140 2400Mass (m/z)

1157.4

0102030405060708090

100

% In

tens

ity

1197.69

1699.82

1345.44

A

A

F

BfB

F

1100 1360 1620 1880 2140 2400Mass (m/z)

3837.9

0102030405060708090

100

% In

tens

ity

1759.88

2283.301345.47

B

BoNT Detection in Multiplexed Reaction by LC-MS/MS

1.2 1.6 2 2.4 2.8 3.2

Inte

nsity

(nor

mal

ized

&

stac

ked)

Time, mins

A

AB

BE

E

F FLEColored by Toxin

Type

Dotted: Internal Stds

LE: Leu enkephalin

High-m/z MRMs

F200

9x11

9x05

_Pep

tide-

Cur

ve_H

igh-

MZ.

wiff

Toxin, amount & matrix BoNT-A (NT) BoNT-B (CT) BoNT-E (NT) BoNT-F (CT)

1 U BoNT-A in serum + - - -

10 U BoNT-A in serum + - - -

1 U BoNT-B in serum - - - -

10 U BoNT-B in serum - + - -

50 U BoNT-E in serum - - + -

500 U BoNT-E in serum - - + -

1 U BoNT-F in serum - - - +

10 U BoNT-F in serum - - - +

BoNT A2b in serum + + - -

Detection of BoNT A, B, E, and F with 4E17.1 Antibody

Multiplexed Detection of BoNT A and B

Max: 1.1e4 cps.A: positive

NT +ve

CT +ve

Max: 5.8e4 cps.B: positive

NT +ve

CT +ve

Max: 467 cps.F: negative

(no peaks)

Max: 733 cps.E: negative

(no peaks)

Summary• Detection of botulinum neurotoxin in clinical samples• Differentiation of the botulinum neurotoxin A

subtypes• Differentiation of two A1 strains of botulinum

neurotoxin• No DNA needed• Accomplished in 8 hr workday• Can be modified to detect in 30 min and subtype in

1 hr• Can be modified to be used on LC-MS/MS

instruments

Acknowledgements

• CDC Mass Spectrometry BoNT Group– Hercules Moura– Wanda I. Santana– Jakub Baudys– Maria I. Solano– Adrian R. Woolfitt– Dongxia Wang– Rebecca Terilli– John R. Barr

• USAMRIID– Leonard A. Smith– Theresa J. Smith

• UCSF– James D. Marks

4E17.1 Epitope on BoNT A1

Protein Sequence Alignment

BoNT/A1(Hall) AIINYQYNQYTEEEKNNINFNIDDBoNT/A2(Honey) AIINYQYNQYTEEEKNNINFNIDDBoNT/A3(Loch Maree) AIINYQYNQYTEEEKNNINFNIDDBoNT/B1(Okra) EIIKYRYNIYSEKEKSNINIDFNDBoNT/B2(213B) EIIKYKYNIYSEKEKSNININFNDBoNT/B3(B17 eukland) EIIKYKYNIYSEEEKSNININFNDBoNT/B4(Bv 657) EIIKYKYNIYSEKERSNINIDFNDBoNT/C1(Brazil) AKIDLEYKKYSGSDKENIKSQVENBoNT/D AKIDLEYKKYSGSDKENIKSQVENBoNT/E1 (Beluga) TIIESKYNSYTLEEKNELTNKYDIBoNT/E2 (544) TIIESKYNSYTLEEKNELTNKYDIBoNT/E3 (Alaska) TIIESKYNSYTLEEKNELTNKYDIBoNT/E4 (Buty Ital) TIIEFKYNSYTLEEKKELKNNYDIBoNT/F (Long) TAIEYKYNNYTSDEKNRLESEYNIBoNT/G KIIEDQYNRYSEEDKMNINIDFNDTetanus Toxin KIIDYEYKIYSGPDKEQIADEINN

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