Multiplex Quantification of Protein Toxins in Human Biofluids and Food Matrices Using Immunoextraction and High-Resolution Targeted Mass Spectrometry

Abstract

The development of rapid methods for unambiguous identification and precise quantification of protein toxins in various matrices is essential for public health surveillance. Nowadays, analytical strategies classically rely on sensitive immunological assays, but mass spectrometry constitutes an attractive complementary approach thanks to direct measurement and protein characterization ability. We developed here an innovative multiplex immuno-LC-MS/MS method for the simultaneous and specific quantification of the three potential biological warfare agents, ricin, staphylococcal enterotoxin B, and epsilon toxin, in complex human biofluids and food matrices. At least 7 peptides were targeted for each toxin (43 peptides in total) with a quadrupole-Orbitrap high-resolution instrument for exquisite detection specificity. Quantification was performed using stable isotope-labeled toxin standards spiked early in the sample. Lower limits of quantification were determined at or close to 1 ng·mL(-1). The whole process was successfully applied to the quantitative analysis of toxins in complex samples such as milk, human urine, and plasma. Finally, we report new data on toxin stability with no evidence of toxin degradation in milk in a 48 h time frame, allowing relevant quantitative toxin analysis for samples collected in this time range.

New Antibody-Free Mass Spectrometry-Based Quantification Reveals That C9ORF72 Long Protein Isoform Is Reduced in the Frontal Cortex of Hexanucleotide-Repeat Expansion Carriers

“…Shorter gradients resulted in interferences at the retention time of peptide DSTGSFVLPFR (SIL version) and a decrease of signal intensity up to a factor of 2 in brain samples ( Supplementary Figure S1 and Supplementary Table S1 ). Data treatment increased sensitivity by summing the signals of up to six major and non-interfered fragment ions to provide one XIC for each targeted peptide (Dupré et al, 2015; Figure 3 and Supplementary Table S2 ). Each endogenous peptide and their corresponding isotope-labeled form (AQUA peptides) must strictly co-elute with similar transition ratio across the different samples in comparison with a standard.…”

Highly sensitive sandwich immunoassay and immunochromatographic test for the detection of Clostridial epsilon toxin in complex matrices

“…There is plethora of scientific reports that address the development of detection tests and counter-measures against diverse potential bioterrorism agents. However, very few specifically document the development of detection tests for epsilon toxin and prototoxin in food or water and diagnostic methods for human body fluids [22,23]. This study describes the development of highly sensitive rapid tests able to address this question.…”

“…The enzyme immunoassays (using either monoclonal or polyclonal antibodies for capture) were nevertheless among the techniques with the highest sensitivity (up to 0.075 mouse lethal dose per mL detected by the polyclonal immunoassay). More recently, a new technique was described using immunopurification followed by liquid chromatography monitored by mass spectrometry [22,23]. To our knowledge, there is only one company (BioX, Belgium) that commercializes kits for the detection of ε toxin.…”

A simple and rapid LC-MS/MS method for therapeutic drug monitoring of cetuximab: a GPCO-UNICANCER proof of concept study in head-and-neck cancer patients

“…Short digestion times were evaluated in combination with modified incubation conditions 14 . A protocol with incubation at 42 °C using 40 µg trypsin was selected as it resulted in robust signal for HT4 and LT3 after 2 hrs and similar intensity to overnight incubation.…”