Species-Specific Discrimination of Insect Meals for Aquafeeds by Direct Comparison of Tandem Mass Spectra

SIMPLE SUMMARY: Aquaculture is amongst the most efficient ways to produce animal protein for human consumption, and this sector is expected to continue to grow worldwide. Inclusion of novel protein sources, like insect meal, may help to mitigate the expected scarcities of feed resources and reduce environmental pressure. However, considered as processed animal protein (PAP), insect meal must comply with the respective legal constraints associated with PAP legislation to guarantee its safety for use as fish feed ingredients. Therefore, there is a need for the development of methods to identify and quantify the species origin of insect-based ingredients in aquafeed. In this study, we propose high-throughput tandem mass spectrometry for the identification and differentiation of 18 different insect meal samples from the species Hermetia illucens (8), Tenebrio molitor (5), Alphitobius diaperinus (3) and Acheta domesticus (2). Using high throughput proteomics tools in combination with direct spectral comparison, we were able to differentiate the insect meal samples according to the taxonomic classification of the insect species. Mass spectrometry-based proteomics is a powerful tool for the species-specific discrimination of insect meals for feed formulations. ABSTRACT: Insect protein has the potential to become a sustainable feed ingredient for the rapidly growing aquaculture industry. In the European Union, insect derived protein is placed under the same legislation as processed animal proteins (PAP). It is therefore of interest to develop methods for regulatory use, which unambiguously identify the species origin of insect-based ingredients. We performed (i) total protein quantification of insect samples using the traditional nitrogen-to-protein conversion factor of 6.25 and the sum of anhydrous amino acids, (ii) quantitative amino acid profiling and (iii) high-throughput tandem mass spectrometry to describe and differentiate 18 different commercial-grade insect meal samples derived from Hermetia illucens (8), Tenebrio molitor (5), Alphitobius diaperinus (3) and Acheta domesticus (2). In addition, we investigated and compared different protein extraction and digestion protocols for proteomic analysis. We found that irrespective of sample preparation, shotgun proteomics in combination with direct spectral comparison were able to differentiate insect meal according to their taxonomic classification. The insect specific spectral libraries created in the present work can in future be used to develop more sensitive targeted methods of insect PAP identification and quantification in commercial feed mixtures.