Safe and effective means of detecting and quantitating Shiga-like toxins in attomole amounts.
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.
Western Regional Research Center, United States Department of Agriculture , Albany, California 94710, United States.
Christopher J
CJ
Silva
Melissa L
ML
Erickson-Beltran
Craig B
CB
Skinner
Irina
I
Dynin
Colleen
C
Hui
Stephanie A
SA
Patfield
John Mark
JM
Carter
Xiaohua
X
He
D000595:Amino Acid Sequence; D000818:Animals; D002470:Cell Survival / Q000187:drug effects; D002522:Chlorocebus aethiops; D002851:Chromatography, High Pressure Liquid; D006868:Hydrolysis; D008969:Molecular Sequence Data; D022622:Shiga Toxin 1 / Q000032:analysis / Q000633:toxicity; D022641:Shiga Toxin 2 / Q000032:analysis / Q000633:toxicity; D022603:Shiga Toxins / Q000032:analysis* / Q000633:toxicity; D014357:Trypsin / Q000737:chemistry; D014709:Vero Cells
D022622:Shiga Toxin 1; D022641:Shiga Toxin 2; D022603:Shiga Toxins; D014357:Trypsin