2005 Fiscal Year Final Research Report Summary
Research and development of new method for forensic chemical detection and identification of high molecular weight biological toxins
| Project/Area Number |
16590103
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Environmental pharmacy
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| Research Institution | National Research Institute of Police Science |
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Principal Investigator |
SETO Yasuo National Research Institute of Police Science, Third Forensic Department, Chief, 法科学第三部, 室長 (10154668)
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| Project Period (FY) |
2004 – 2005
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| Keywords | biological toxin / forensic chemistry / detection and identification / mass spectrometry / protein / on-site detection / preliminary test |
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| Research Abstract |
The forensic chemical methodology has been established for detecting and identifying high molecular weight biological toxins such as plant toxin ricin, from on-site detection to laboratory preliminary and identification analysis.
(1)On-site detection
Detection limits of immunostrip BTA system against ricin, Staphylococcal enterotoxin (SEB) and botulinum toxins were ranging about 0.1 μg/ml, and addition of white powder materials and colored matrices did not disturb the detection. Sample heating (above 80℃) and addition of hypochlorite (above 0.001%) and formaldehyde (above 0.3 M) led to the false negative results. Surface plasmon resonance analysis using gold sensor plate attached of lactose binding ceramide derivatives gave sensitive detection against ricin (about 10 ng/ml).
(2)Preliminary laboratory analysis
SDS-Polyacrylamide gel electrophoresis (8% gel) gave two bands (31-32 kDa) and one band (60 kDa) against ricin with and without β-mercaptoethanol pretreatment.
(3)Mass spectrometry
MALD
… More
I-TOF-MS analysis gave broad molecular ion peaks around m/z 28425 and 62594 against SEB and ricin under the conditions of sinapinic acid matrix and linear ion mode. LC-MS analysis gave late-eluting broad band against SEB with multiply charged ions centered at m/z 836, resulting in MW 28364 with deconvolution analysis, and gave late-eluting broad tailing band against ricin with multiply charged ions centered at m/z 1000, under the conditions of large-porous ODS capillary column, formic acid-acetonitrile elution and electrospray ionization.
(4)Digestion LC-MS
SEB and ricin were treated with guanidine, dithiothreitol and iodoacetate, trypsinized after desalting, and analyzed by LC-MS with the conditions of large-porous ODS capillary column, formic acid-acetonitrile elution and electrospray ionization. On liquid chromatograms, multiple digestion peaks appeared, and 10 (SEB) and 13 (ricin) peptide peaks were structurally confirmed by MS/MS with Mascot analysis. Detection limits were 3 (SEB) and 6 (ricin) μg/ml. Less
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