Rapid Detection Of Conotoxin SO3 In Serum Using Cu Chelated Magnetic Beads Coupled With MALDI-TOF-MS

Rapid and accurate detection and identification of specific biological toxins is vital for clinical diagnostics, public health safety, and biological warfare defense. The potential use of biological toxins as weapons of mass destruction has created an urgent need for rapid and highly sensitive assays for their detection. Methods to detect them include mouse bioassays, immunoassays, biosensors and genetic analysis. Mouse bioassays are very labor-intensive and need large numbers of animals. Nucleic acid-based genetic analysis, e.g., polymerase chain reaction (PCR), involves DNA amplification that offers high sensitivity and unambiguous identification. However, it is not applicable for the detection of toxins that contain no nucleic acids. Immunoassays are advantageous over other methods for their simplicity and rapidness, but require specific antibodies to the agent of interest. Therefore, they cannot be used for detecting unknown or new agents because a single antibody is unavailable.Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) is a soft ionization method and has become a rapid and convenient method for the characterization of biomolecules such as proteins and peptides in various fields. Scientists have developed methods based on MALDI-TOF-MS to detect biological toxins in different media, such as water, food, and the environment due to their high sensitivity, tolerance to impurities, and high speed. However, few have focused on biological fluids such as serum, because serum is a very complex mixture of proteins, peptides, and small molecules, with a concentration range spanning over 10 orders of magnitude. Suppression effects and interference of other biomolecules made serum analysis unsuitable for direct assay by MALDI-TOF-MS.Since the mid-1970s, there has been increasing development of magnetic beads for applications in bioscience and medicine. Extensive applications of magnetic beads in separation of analytes from complex media have been well documented. Recently, proteomic methods for serum peptide profiling have been developed based on magnetic beads and MALDI-TOF-MS. The ability of magnetic beads to separate and enrich peptides from serum made it possible to detect peptide toxins when coupled with MALDI-TOF-MS.In this article, a novel method based on Cu chelated magnetic beads (Cu-Magbeads) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was developed for the rapid detection of peptide toxins in serum. The peptides in the serum were efficiently adsorbed by the Cu-Magbeads, eluted with methanol solution, and assayed by MALDI-TOF-MS. Specific peptides were identified according to their characteristic m/z values. Conotoxin SO3, a synthesized peptide, was used as a model to evaluate the method. Conotoxin SO3 was detected in human serum, as well as in bovine and murine serum, with a detection sensitivity in the low femtomole level. The assay was performed within 40 min, without the need for a specific antibody or an expensive reagent. It shows potential for future use in clinical and emergency rescue practice due to the low cost, high speed, and high sensitivity.