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
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
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
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
BoNT extraction 1 hr
Endopep-MS Reaction 4 hrs
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
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
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
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
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
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
BoNT extraction 1 hr
Endopep-MS Reaction 4 hrs
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