Safe and Effective Means of Detecting and Quantitating Shiga-Like Toxins in Attomole Amounts
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- 作者：Christopher J. Silva ; Melissa L. Erickson-Beltran ; Craig B. Skinner ; Irina Dynin ; Colleen Hui ; Stephanie A. Patfield ; John Mark Carter ; Xiaohua He
- 刊名：Analytical Chemistry
- 出版年：2014
- 出版时间：May 20, 2014
- 年：2014
- 卷：86
- 期：10
- 页码：4698-4706
- 全文大小：305K
- 年卷期：v.86,no.10(May 20, 2014)
- ISSN：1520-6882
文摘
Shiga-like toxins (verotoxins) are a class of AB5 holotoxins that are primarily responsible for the virulence associated with Shiga-like toxin producing Escherichia coli (STEC) infections. The holotoxins are composed of a pentamer of identical subunits (B subunit) responsible for delivering the catalytic subunit (A subunit) to a host cell and facilitating endocytosis of the toxin into the cell. The B subunits are not associated with toxicity. We developed a multiple reaction monitoring method based on analyzing conserved peptides, derived from the tryptic digestion of the B subunits. Stable-isotope-labeled analogues were prepared and used as internal standards to identify and quantify these characteristic peptides. We were able to detect and quantify Shiga toxins (Stx), Shiga-like toxin type 1 (Stx1) and type 2 (Stx2) subtypes, and to distinguish among most of the known subtypes. The limit of detection for digested pure standards was in the low attomole range/injection (10 attomoles), which corresponded to a concentration of 1.7 femtomol/mL. A matrix effect was observed when dilute samples were digested in the buffer, Luria broth, or mouse plasma (LOD 30 attomol/injection = 5 femtomol/mL). In addition, we determined that the procedures necessary to perform our mass spectrometry-based analysis completely inactivate the toxins present in the sample. This is a safe and effective method of detecting and quantitating Stx, Stx1, and Stx2, since it does not require the use of intact toxins.