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

Abstract

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 100folds. 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.

Keywords: Biomarkers; Fish muscle tissue exudates; Magnetic beads (MBs); Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS); Species-specific peak markers.

Graphical abstract

Fig. 1

(A) Representative MALDI-TOF mass spectra for fish exudate samples of NA after protein extraction by using WCX-MBs in different pH environments. The pH of the utilized buffer was labeled as inlets. (B) Representative MALDI-TOF mass spectra for fish exudate samples of NA after protein extraction by using WCX magnetic beads after diluting the exudates with sodium citrate buffer (50 mM, pH 5.0). The fold of dilution is labeled as inlets. The MBs pellets were added into diluted exudate sample. The mixture was shaken to extract proteins in exudate samples, then magnetically separated and the pellets were collected. 10 μL of TFA solution (1%, v/v) was added to elute the proteins on MBs pellets for further analysis by MALDI-TOF MS. (C) Representative MALDI-TOF mass spectra of muscle tissue exudates from five fish species, NA, LC, MM, CL, and LP under optimized conditions. (D) Representative MALDI-TOF mass spectra for tryptic digests of muscle tissue exudates from five fish species, NA, LC, MM, CL, and LP under optimized conditions. (E) Representative MALDI-TOF mass spectra of muscle tissue exudates from five fish species, NA, LC, MM, CL, and LP with enrichment by WCX-MBs (red) under optimized conditions. (F) Representative MALDI-TOF mass spectra for tryptic digests of muscle tissue exudates from five fish species, NA, LC, MM, CL, and LP with enrichment by WCX-MBs (red) under optimized conditions. The peak m/z values of potential markers are denoted in the diagram. The m/z of the peptides that match the theoretically predicted peptide masses are also denoted in the diagram. The black color labeled peaks in (C) to (F) are the identified mass spectral peak markers. The other colored peaks are the identified biomarkers and their corresponding digested peptides. The proteins on MBs pellets were eluted and digested for analysis by MALDI-TOF MS. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

(A) Plot of PCA scores for MALDI-TOF mass spectra of muscle exudates that flowed from five analyzed fish species. (B) Plot of VIP scores for MALDI-TOF mass spectra of muscle exudates collected from five analyzed fish species. Experimentally, the collected exudate samples were diluted 100 folds with DI water prior to MALDI-TOF MS analysis. (C) Plot of PCA scores for MALDI-TOF mass spectra of muscle exudates collected from five analyzed fish species under enrichment of WCX-MBs. (D) Plot of VIP scores for MALDI-TOF mass spectra of muscle exudates collected from five analyzed fish species under enrichment of WCX-MBs. The collected exudate samples were enriched with WCX-MBs after diluting the exudates 100-folds with sodium citrate buffer (50 mM, pH 5.0). (E) MALDI-TOF mass spectra for unknown sample (US) 1 and 2 and their tryptic digests for proteins. The proteins were extracted from fish exudate samples from unknown samples, US1 and US2, via WCX-MBs. Fish muscle exudates of two fish samples randomly selected from the studied fishes were collected and analyzed as single-blind samples.