LC/QTOF Identification of Microcystin Variants in Cell- Bound Algae from the San Francisco Estuary Ralph Hindle Vogon Laboratory Services Ltd. Cochrane, Alberta, Canada Max Mizel Dept. of Civil and Environmental Engineering UC Davis Davis, California 1
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LC/QTOF Identification of
Microcystin Variants in Cell-
Bound Algae from the San
Francisco Estuary
Ralph Hindle
Vogon Laboratory Services Ltd.
Cochrane, Alberta, Canada
Max Mizel
Dept. of Civil and Environmental Engineering
UC Davis
Davis, California
1
• Investigate impact of Microcystis aeruginosa in
San Francisco Estuary Delta in summer/fall of
2014
• Magnitude
• Timing
• Distribution
• Toxin content (cell-bound)
• Identify and quantitate MC-LR
• Identify other MC’s that may be present
• Nitrogen source
• Food web impact
Project Outline
Sampling Sites
3
General Microcystin Structure
> 70 microcystins have been identified in the literature
Microcystin
Examples
X Z Formula Neutral Mass
LR Leucine Arginine C49H74N10O12 994.5488
Desmethyl LR Leucine Arginine C48H72N10O12 980.5331
RR Arginine Arginine C49H75N13O12 1037.5658
YR Tyrosine Arginine C52H72N10O13 1044.5280
LW Leucine Phenylalanine C54H72N8O12 1024.5270
LF Leucine Tryptophan C52H71N7O12 985.5161
HtyR Homotyrosine Arginine C53H74N10O13 1058.5437
QQQ MRM Parametersa
5
a These patterns are useful, as they show that all supplied MCs fragment to the same 2 diagnostic ions
b RR readily shows a doubly-charged precursors. Depending on source conditions, other MCs can show either singly- or doubly-charged ions.
Microcystin Precursor b
(m/z)
Product Ion
(m/z)
Collision Energy
(V)
LR 995.6 135.2
213.2
80
80
Desmethyl-LR 981.5 135.2
213.2
80
80
RR 520.0 135.2
213.2
30
40
YR 1045.5 135.2
213.2
80
70
LW 1025.5 135.2
213.2
80
60
LF 986.5 135.2
213.2
70
50
HtyR 1059.5 135.2
213.2
80
70
Common Microcystin Fragments
m/z = 135.1 and 213.1
C9H11O+
m/z = 135.0804
Glu + Mdha = C9H13N2O4+
m/z = 213.0870
Zoom in on common MC fragments - 135
135.0804 = C9H11O+
• De-methylation is common in microcystins• Two 135 ions are present from 2 different
fragments• Indicates that Adda group is intact and not
de-methylated
135.1168 = C10H15+
C9H11O+
C10H15+
LC-QTOF Instrumentation
8
HPLC Mass Spectrometer
1290 Binary Pump 6520 or 6540 QTOF
1200 Degasser Electrospray Source
1290 Autosampler
1290 TCC
Database Score
Monoisotopic mass(varies in ppm)
Isotope spacing(varies in ppm)
Isotope distribution(varies in %)
Score based on:
• Mass match – measured versus given.
• Abundance score – abundance pattern of measured isotope cluster compared withvalues predicted from the proposed formula.
• Spacing match – how the m/z spacingbetween the lowest m/z ionand the A+1 and A+2 ions compared withthe values predicted from the proposed formula.
Chromatography
Mass tolerance 5 ppm; EIC extraction 50 ppm; spectral average < 10% saturation
12
Product Ion Spectrum for MC-LR
Score = 97.7Acc = 1.9 ppm
13
Product Ion Spectrum for MC-RR
Score = 96.6Acc = 2.3 ppm
14
Product Ion Spectrum for MC-LA
Score = 95.0Acc = 1.0 ppm
MC-LR Desmethylation Sites
Arg4
Leu2
MeAsp3
Ala1
Mdha7Glu6
Adda5
C49H74N10O12
Mass = 994.5488C48H72N10O12
Mass = 980.5331
- CH2
- 14.0157
Comparison of IC50 for MC Variants
17
MC Variant IC50 (µg/mL)
[Asp3, Z-Dhb7] MC-LR 0.05
[Asp3, Z-Dhb7] MC-HtyR 0.12
[Asp3, E-Dhb7] MC-LR 0.13
[Asp3, Dha7] MC-LR 0.22
[Asp3] MC-LR 0.22
[Dha7] MC-LR 0.22
[Asp3, E-Dhb7] MC-HtyR 0.33
[Asp3] MC-HtyR 0.35
[Dha7] MC-YR 0.42
MC-LR 0.80
MC-YR 1.48
[Asp3, Dha7] MC-RR 4.11
[Asp3, E-Dhb7] MC-RR 4.95
[Dha7] MC-RR 5.33
[Asp3] MC-YR > 10
MC-RR > 10
IC50 = Inhibitory Concentration for cytotoxicity, from Shimizu et al, Toxins 2014, 6, 168-179
Trp200 His191
Gln122
Val126Tyr127Tyr265
Cys269
Arg89
Arg214
Ile123
Glu
53
X-ray crystal structures of MC-LR bound to the catalytic domain of PP2A. The data is from the protein database on the net, provided as part of Xing, Y., Xu, Y., Chen, Y., Jeffrey, P. D., Chao, Y., Lin, Z., Li, Z., Strack, S., Stock, J. B., and Shi, Y. (2006) Structure of protein phosphatase 2A core enzyme bound to tumor-inducing toxins. Cell 127, 341–353. The picture was generated by Christopher Miles (Oslo, Norway) using Pymol.