Analysis of Biomarker Peptides and Allergen Specific Peptide in Salmo Salar by Proteomics Based on Biomass Spectrometry

Based on the bottom-up research strategy of proteomics, nano liquid chromatography-quadrupole-electrostatic field orbital trap high resolution mass spectrometry (Nano-UHPLC-Q/Exactive-HRMS) and ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) were used to screen and identify biomarker peptides and allergen β-parvalbumin specific peptide in Salmo salar, and a method for the determination of them by UHPLC-MS/MS was proposed. The processed sample (5 g) was taken, and 10 mL of n-hexane was added for grease removal. After centrifugation, 5 mL of phosphate buffer solution (as extractant) was added into the precipitation. The mixture was whirled and heated in water bath at 60 ℃ for 30 min, and 20 mL of cold acetone was used for enrichment and purification. The obtained protein extract was hydrolyzed by trypsin, and Nano-UHPLC-Q/Exactive HRMS was used to preliminary screen the biomarker peptides and the specific peptide of allergen β-parvalbumin in Salmo salar, and then UHPLC-MS/MS was used to verify the screened peptides with multiple reaction monitoring (MRM) mode. Internal standards were added into the solution after enzymolysis, and UHPLC-MS/MS was used for determination, with internal standard method for quantitative analysis. It was shown that 3 biomarker peptides VAVNVEETK, VAPLSEDFK, LTEIQSLER and one allergen β-parvalbumin specific peptide TFFHTIGFASK were screened for quantification. The linear ranges of standard curves for VAVNVEETK, VAPLSEDFK and LTEIQSLER were within 5.00 μmol·L^-1, and that for TFFHTIGFASK was within 10.00 μmol·L^-1, with detection limits (3S/N) in the range of 0.32-1.55 μg·g^-1. The recoveries of 4 peptides in the blank samples ranged from 90.2% to 105%, and RSDs (n=6) of the determined values were in the range of 1.5%-4.6%. This method was applied to determination of VAVNVEETK, VAPLSEDFK, LTEIQSLER and TFFHTIGFASK during the storage of Salmo salar. The contents of LTEIQSLER and TFFHTIGFASK did not change significantly, the contents of VAVNVEETK and VAPLSEDFK decreased gradually with the extension of storage days, and VAPLSEDFK was suitable for the freshness evaluation of Salmo salar.