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Proteomic analysis of human follicular fluid: A new perspective towards understanding folliculogenesis The poster is designed and prsented by Ankur Tyagi Guided by: Dr. Arun A. B. Yenepoya Research Centre, Yenepoya University , Mangalore This paper is cited from Aditi S. Ambekar, Raja Sekhar Nirujogi, Srinivas M. Srikanth, Sandip Chavan, Dhanashree S. Kelkar, Indira Hinduja , et. al. Proteomic analysis of human follicular fluid: A new perspective towards understanding folliculogenesis J O U R N A L O F P R O T E O M I C S. 2 0 1 3; 8 7: 6 8 – 77. Introduction Follicular fluid is a vital and dynamic element of the ovarian follicle for developing oocyte. The accumulation of follicular fluid in the growing follicle begins at the preantral stage by diffusion of proteins in the blood through the calcapillaries and secretions from granulosa cells (GCs), theca cells and oocytes. The protein content of follicular fluid reflects changes in the secretory processes of the GCs, theca cells and their metabolic status during abnormal follicular growth. Proteomic profile of human follicular fluid in reproductive disorders including recurrent spontaneous abortion, endometriosis, ovarian hyperstimulation syndrome (OHSS) and poor responders to IVF provided some information on their pathophysiology Materials and methods 1. Sample collection: Follicular fluid (~5 ml) was obtained from six regularly menstruating healthy women undergoing IVF due to male factor infertility from INKUS IVF Clinic (Mumbai, India). This work has been approved by the Institutional Ethics Committee of NIRRH. 2. Depletion of high abundance proteins: Protein estimation was carried out by Lowry's method. We pooled equal amounts of protein from six samples. Pooled sample (52 mg/ml protein concentration) was diluted six times in buffer A (p. H 7. 4) (Agilent Technologies) and then subjected to Human 14 Multiple Affinity Removal System (MARS-14) liquid chromatography (LC) (Agilent Technologies, Santa Clara, USA). 3. SDS-PAGE and in-gel digestion: Approximately 250 μg of protein was resolved on a 10% SDS-PAGE gel. Protein bands were destained and subjected to reduction and alkylation. 4. In-solution digestion: Approximately 500 μg protein was subjected to reduction, alkylation and tryptic digestion. The digested peptides were divided equally in two parts and subjected to OFFGEL fractionation and SCX. I. OFFGEL fractionation II. Strong cation exchange chromatography 5. LC–MS/MS analysis: Tandem mass spectrometric analyses of all the 66 fractions were carried out using an LTQ-Orbitrap Velos mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) interfaced with Easy-n. LC II nanoflow liquid chromatography system (Thermo Scientific, Odense, Denmark). 6. Data analysis: Peptide identification was carried out using both SEQUEST (SCM build 59) and MASCOT (version 2. 2) search engines to improve the peptide identification. Proteome Discoverer 1. 3 (Thermo Fisher Scientific, Bremen, Germany) software was used to generate the peak list Results Table 1: List of the angiogenic factor identified in follicular fluid Fig 1: Representative MS/MS spectra of selected peptides from a subset of proteins identified from follicular fluid Distribution of proteins identified from follicular fluid using the three techniques employed for protein/peptide fractionation, Classification of follicular fluid proteins (A) subcellular localization (B) molecular function. Conclusion This data will contribute to the better understanding of follicular fluid function and to the discovery of potential biomarker for oocyte quality, pregnancy outcome and probably investigating the infertility issues in the future. Acknowledgement I hereby acknowledge Yenepoya research centre for their help. I would like to thank Yenepoya University for the fellowship.