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  • 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 M ZnCl2 , and 1 mg/mL BSAReaction volume is 20 LAfter incubation, reaction is diluted 1:10 in matrix solution, 5 mg/mL HCCA

  • Detection of BoNT Activity

    Enzymatic reaction mixed with matrix compoundMixture spotted onto stainless steel MALDI plate and allowed to dryPlate analyzed by MALDI-TOF/MS on Applied Biosystems 4800 Proteomics AnalyzerAnalysis is 15 seconds per sample

  • Detection of BoNT Activity

    Enzymatic reaction mixed with matrix compoundMixture spotted onto stainless steel MALDI plate and allowed to dryPlate analyzed by MALDI-TOF/MS on Applied Biosystems 4800 Proteomics AnalyzerAnalysis is 15 seconds per sample

    m/z

    IntAbsence of BoNT

  • Detection of BoNT Activity

    Enzymatic reaction mixed with matrix compoundMixture spotted onto stainless steel MALDI plate and allowed to dryPlate analyzed by MALDI-TOF/MS on Applied Biosystems 4800 Proteomics AnalyzerAnalysis 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 AA1A2A3A4

    BoNT BB1B2bivalent BNp B

    BoNT EE1E2E3E It butyricumE Ch butyricum

    BoNT Fprot Fnp Fbivalent FF baratii

    BoNT CC1Mosaic C

    BoNT DD1Mosaic D

    BoNT GG1

  • LOD in serumBoNT 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 serumBoNT 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 extractBoNT 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 SamplesBoNT 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

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