Discovery of mass spectral peak markers and protein biomarkers in fish muscle exudates for rapid and precise recognition of fish species via magnetic beads (MBs) and mass spectrometry

In this study, muscle exudates from five fishes belonging to the family Sciaenidae, in the order Perciformes, were analyzed as models for the discovery of biomarkers by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). MagSi-weak cation exchange magnetic beads (WCX-MBs) were utilized for the enrichment of proteins from fish exudate samples, allowing protein biomarkers to be identified and subsequently used for fish species differentiation. Buffers with pH ranging from 4.0 to 9.0 can provide an environment for proteins in fish muscle exudate to bind to the WCX-MBs. The optimal enrichment based on WCX-MBs can be achieved when the exudate samples are diluted 100-folds. More species-specific biomarkers in mass spectra can be identified when using WCX-MBs. The number of ions that can be considered as peak markers and can differentiate the analyzed fishes increases from 38 to 121 when using WCX-MBs to isolate peptides/protein in fish muscle exudate. Particularly, eight peak markers in mass spectra were assigned to be specific to Nibea albiflora (NA), three peak markers specific to Larimichthys crocea (LC), two peak markers specific to Miichthys miiuy (MM), seven peak markers specific to Collichthys lucidus (CL), and six peak markers specific to Larimichthys polyactis (LP). Furthermore, five proteins were identified based on the characterization of tryptic peptides and their potential to be biomarkers, of which four proteins specific to CL and one specific to LC were identified. The single-blind samples analysis demonstrated that these species-specific peak markers and protein biomarkers can be successfully utilized for corresponding fish recognition. The utilization of WCX-MBs can improve the discovery of fish species-specific biomarkers in fish muscle exudate samples. The present protocol holds potential of being a rapid and accurate identification tool for recognition of fish species.