Compatibility of distinct label-free proteomic workflows in absolute quantification of proteins linked to the oocyte quality in human follicular fluid (the Quad-Orbitrap workflow)

We present a comprehensive analysis of human follicular fluid proteome by comparing several strategies of sample preparation, mass spectrometry analysis, and data processing, resulting in the identification of 1638 proteins. We devised two separate label-free quantitative workflows, termed after the utilized mass spectrometer: Triple Quad-TOF and Quad-Orbitrap, and analyzed their performance in absolute protein quantification assisted by Total Protein Approach. In a pilot study of twenty samples linked to diverse oocyte quality status from four donors, 215 and 455 proteins were quantified by the Triple Quad-TOF and Quad-Orbitrap workflows, respectively. The concentration values obtained from both workflows correlated to a significant degree. We found reasonable agreement of both workflows in protein fold changes between tested groups, resulting in unified lists of 21 and 24 proteins, linked to oocyte maturity and blastocyst development, respectively. The Quad-Orbitrap workflow was best suited for an in-depth analysis, especially of low abundant proteome, whereas the Triple Quad-TOF workflow allowed a more robust approach with a greater potential to increase in effectiveness with the growing number of analyzed samples.