On-Line Solid Phase Extraction Liquid Chromatography-Mass Spectrometry Method for Multiplexed Proteins Quantitation in an Ecotoxicology Test Specie: Gammarus fossarum

摘要

Objectives A fully automated on-line solid phase extraction procedure was developed and validated for the analysis of 30 key proteins biomarkers in Gammarus fossarum. Methods After protein extraction and tryptic digestion, peptides were cleaned-up onto an on-line SPE cartridge (Oasis HLB, 2.1 mm x 20 mm, 25 μm particle size) coupled to a LC-MS/MS system. The SRM assay was performed on a quadrupole-trap mass spectrometer. Results The method targeted 30 proteins in G. fossarum (46 reporter peptides used in the SRM assay) was developed and validated. The method duration was 30 min including the on-line SPE step saving up to 6h per sample. The method’s performance was validated according to FDA guidelines Conclusions Our method substitutes valuably conventional methods like off-line SPE performed beforehand to the LC-MS/MS system. This assay offers higher sensitivity with no loss and/or degradation of reporter peptides and reached good specifications (linearity, precision and accuracy).