The data acquired by the iTRAQ analysis were further verified by quantifying the abundance of 6 selected proteins using PRM. The signature peptides for the target proteins were defined according to the iTRAQ data and unique peptide sequences were selected for the PRM analysis. PRM analysis was carried out on the Q-Exactive HF spectrometer (Thermo, USA). The raw data obtained were analyzed using the Proteome Discoverer and the FDR was set at 1% for peptides. The resulting MS data were processed by using Skyline (version 3.5.0, RRID: SCR_014080).
PRDX6 antibody (Abmart, Cat No T56784, China) was used to quantify the PRDX6 abundance of GFE and PFE sperms, and β-Tublin antibody (Abmart, Cat No M30109, RRID: AB_2916070) was used as control. Goat anti-rabbit mouse IgG-HRP was used as the secondary antibody (Abmart, Cat No M21003, RRID: AB_2920649). Western blot was performed as described previously [13 (link)]. Considering the antibodies used were unspecific for buffalo, we cut the membrane prior to hybridization to remove the nonspecific blots. Quantification employed ImageJ (NIH Image J system, USA, RRID: SCR_003070) and the data were normalized to β-Tublin. Each loading sample for WB analysis consisted of three mixed ejaculates.
PRDX6 antibody (Abmart, Cat No T56784, China) was used to quantify the PRDX6 abundance of GFE and PFE sperms, and β-Tublin antibody (Abmart, Cat No M30109, RRID: AB_2916070) was used as control. Goat anti-rabbit mouse IgG-HRP was used as the secondary antibody (Abmart, Cat No M21003, RRID: AB_2920649). Western blot was performed as described previously [13 (link)]. Considering the antibodies used were unspecific for buffalo, we cut the membrane prior to hybridization to remove the nonspecific blots. Quantification employed ImageJ (NIH Image J system, USA, RRID: SCR_003070) and the data were normalized to β-Tublin. Each loading sample for WB analysis consisted of three mixed ejaculates.
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