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. 2021 Oct 25;11:20979. doi: 10.1038/s41598-021-00494-1

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© The Author(s) 2021

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Experimental design. Human spermatozoa were capacitated for 4 h in the presence/absence of a NO donor (GSNO) and two NOS inhibitors (l-NAME and AG). The experimental groups were supplemented or not with L-Arginine and/or follicular fluid (FF). Sperm proteins were subjected to electrophoresis followed by Western Blot to analyze the phosphorylation levels of phospho-PKA substrates (PKAs-P) and tyrosine residues (Tyr-P). The amount of signal in each membrane was determined by chemiluminescence and, subsequently, quantified. Specific protein bands, that showed significant differences amongst the treatments mentioned above, were subjected to in-gel trypsin digestion, followed by mass spectrometry analysis (HPLC-ESI-Q-TOF–MS/MS). Testis-specific protein–protein interaction and fertility-related knockout phenotypes were also analyzed.