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

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. : After protein extraction and tryptic digestion, peptides were cleaned-up onto an on-line SPE cartridge (Oasis HLB, 2.1 mmx20 mm, 25 μm particle size) coupled to a LC-MS/MS system. The SRM assay was performed on a quadrupole-trap mass spectrometer. : 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. : 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).