Matrix-assisted laser-desorption and ionization time-of-flight mass spectrometry prepares proteins intact in the gas phase with predominantly a single positive charge. The times-of-flight of charged proteins along a tube held at high vacuum after acceleration in an electrical field are proportional to the square root of the mass-over-charge ratios for the proteins, thereby allowing a mass spectrum to be generated, which can then be used to characterize or identify a protein-containing sample. Several sample-preparation methods are currently available but not all of these are applicable to some forms of fungal biomass and few of these are well suited to the analysis of plant or insect material. We have therefore developed a simplified method that: lyses cells, selectively solubilizes basic proteins, dissolves matrix to a suitable concentration, generates spectra with good intensity and peak richness, costs no more (and generally less) than current methods, and is not constrained in terms of throughput by the availability of centrifuges. Using this method, and a reagent formulation comprising α-cyano-4-hydroxycinnamic acid matrix close to saturation in 60%-65% (v/v) acetonitrile in water containing 2.5% (v/v) trifluoroacetic acid, we have been able to differentiate between strains for a representative subset of aflatoxin-producing and aflatoxin-non-producing strains of Aspergillus fungi, to differentiate between Indian and Pakistani strains of Himalayan balsam rust, to differentiate between closely-related Crassula spp. and regional biotypes of Crassula helmsii, and to differentiate between rubbervine introduced into Australia and Brazil. We have also analyzed fall armyworm and stem-borer samples stored in 70% (v/v) ethanol and old dried insect specimens.
Keywords: reagent optimization; sample-preparation; species discrimination; subspecies discrimination.