Abstract
Protamines replace histones as the main nuclear protein in the sperm cells of many species and play a crucial role in compacting the paternal genome. Human spermatozoa contain protamine 1 (P1) and the family of protamine 2 (P2) proteins. Alterations in protamine PTMs or the P1/P2 ratio may be associated with male infertility. Top-down proteomics enables large-scale analysis of intact proteoforms derived from alternative splicing, missense or nonsense genetic variants or PTMs. In contrast to current gold standard techniques, top-down proteomics permits a more in-depth analysis of protamine PTMs and proteoforms, thereby opening up new perspectives to unravel their impact on male fertility. We report on the analysis of two normozoospermic semen samples by top-down proteomics. We discuss the difficulties encountered with the data analysis and propose solutions as this step is one of the current bottlenecks in top-down proteomics with the bioinformatics tools currently available. Our strategy for the data analysis combines two software packages, ProSight PD (PS) and TopPIC suite (TP), with a clustering algorithm to decipher protamine proteoforms. We identified up to 32 protamine proteoforms at different levels of characterization. This in-depth analysis of the protamine proteoform landscape of normozoospermic individuals represents the first step towards the future study of sperm pathological conditions opening up the potential personalized diagnosis of male infertility.
Highlights
Protamines are the main nuclear proteins in the sperm cells of many species, and they play a crucial role in the correct packaging of the paternal DNA [1,2,3]
We suggested that the presence of human sperm protamine proteoforms, including truncations or proteoforms containing post-translational modifications (PTMs), explain the variability observed in the protamine 1 (P1)/protamine 2 (P2) ratio assessed by conventional polyacrylamide gel electrophoresis (PAGE) [30,31]
The results presented here are all the data combined with the final aim of characterizing the protamine proteoforms in the samples
Summary
Protamines are the main nuclear proteins in the sperm cells of many species, and they play a crucial role in the correct packaging of the paternal DNA [1,2,3]. During the final stage of spermatogenesis (called spermiogenesis), the chromatin of immature germ cells undergoes marked remodeling, in which histones are sequentially replaced by specific histone variants, transition proteins and, protamines [1,4,5,6,7]. Both the small size of protamines (20 to 55 amino acids) and their extremely rich sequence in positively charged arginine residues allow the formation of highly condensed toroidal complexes, together with the negatively charged paternal DNA [8,9]. This distinctive chromatin structure has been proposed as a specific epigenetic mark of sperm that could regulate gene expression once the oocyte has been fertilized [19,20,21]
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