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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)

ABSTRACT: 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.

INSTRUMENT(S): Q Exactive HF-X

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Follicular Fluid

SUBMITTER: Aleksandra Bogucka

LAB HEAD: Jacek R Wisniewski

PROVIDER: PXD024347 | Pride | 2021-07-23

REPOSITORIES: Pride

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			Action	DRS
	20180411_QX8_JRW_SA_CT_GG112.raw	Raw
	20180411_QX8_JRW_SA_CT_GG113.raw	Raw
	20180411_QX8_JRW_SA_CT_GG114.raw	Raw
	20180411_QX8_JRW_SA_CT_GG115_180628151102.raw	Raw
	20180411_QX8_JRW_SA_CT_GG116.raw	Raw

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Project description:In mammals, the capacity of the female germ cell, the oocyte, to develop into embryo is acquired throughout meiotic maturation. Immature oocyte cannot be fertilized while mature oocyte is apt to accept spermatozoa and to develop an embryo. In a follicle, the oocyte is surrounded by mural granulosa cells (GC) and is physically and metabolically coupled with specialized granulosa cumulus cells (CC) which play an important role in oocyte maturation and fertilization. Factors expressing in GC and CC during maturation may reflect the oocyte quality, i.e. its capacity to be fertilized and assure early embryo development. However, the modifications of the content and the amount of peptide/proteins in the oocyte and the surrounding CC during oocyte maturation are mostly unknown and so there is not an accurate way of evaluating/monitoring how different in vitro maturation (IVM) protocols being in use in assisted reproduction technologies, can affect the process. In this context, Intact Cell MALDI-TOF Mass Spectrometry (ICM-MS) was applied to bovine follicular cells (bovine single oocytes, cumulus cells and granulosa cells) in order to characterize proteomic changes that occur in the follicle during female gamete development. In order to characterize finely endogenous molecular species observed on ICM-MS profiles and to identify markers of interest with their post-translational modifications, we carried out top-down proteomic on the different follicular cells from oocyte, oocyte-cumulus complexes, cumulus cells and granulosa cells protein extracts. Prior to top-down MS using a dual linear ion trap Fourier Transform Mass Spectrometer LTQ Orbitrap Velos, depending on the amount of available biological material, we employed three analytic strategies as a direct infusion, a mono-dimensional liquid chromatography (µLC-1D-MS/MS) and an off-line multi-dimensional liquid chromatography combining four fractionations (based on reverse phase or gel filtration LC) to µLC-MS/MS. Here, we deposited our dataset from µLC-1D-MS/MS (analyses of oocytes, oocyte-cumulus complexes, cumulus cells and granulosa cells protein extracts) and MDLC-MS/MS (analyses of granulosa cells protein extract).

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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)

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