Protamine Characterization by Top-Down Proteomics: Boosting Proteoform Identification with DBSCAN
Gianluca Arauz-Garofalo,
Meritxell Jodar,
Mar Vilanova,
Alberto de la Iglesia Rodriguez,
Judit Castillo,
Ada Soler-Ventura,
Rafael Oliva,
Marta Vilaseca,
Marina Gay
Affiliations
Gianluca Arauz-Garofalo
Institute for Research in Biomedicine (IRB Barcelona), The Barcelona Institute of Science and Technology, Baldiri Reixac, 10, 08028 Barcelona, Spain
Meritxell Jodar
Molecular Biology of Reproduction and Development Research Group, Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Fundació Clínic per a la Recerca Biomèdica (FCRB), Faculty of Medicine and Health Sciences, University of Barcelona, 08036 Barcelona, Spain
Mar Vilanova
Institute for Research in Biomedicine (IRB Barcelona), The Barcelona Institute of Science and Technology, Baldiri Reixac, 10, 08028 Barcelona, Spain
Alberto de la Iglesia Rodriguez
Molecular Biology of Reproduction and Development Research Group, Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Fundació Clínic per a la Recerca Biomèdica (FCRB), Faculty of Medicine and Health Sciences, University of Barcelona, 08036 Barcelona, Spain
Judit Castillo
Molecular Biology of Reproduction and Development Research Group, Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Fundació Clínic per a la Recerca Biomèdica (FCRB), Faculty of Medicine and Health Sciences, University of Barcelona, 08036 Barcelona, Spain
Ada Soler-Ventura
Molecular Biology of Reproduction and Development Research Group, Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Fundació Clínic per a la Recerca Biomèdica (FCRB), Faculty of Medicine and Health Sciences, University of Barcelona, 08036 Barcelona, Spain
Rafael Oliva
Molecular Biology of Reproduction and Development Research Group, Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Fundació Clínic per a la Recerca Biomèdica (FCRB), Faculty of Medicine and Health Sciences, University of Barcelona, 08036 Barcelona, Spain
Marta Vilaseca
Institute for Research in Biomedicine (IRB Barcelona), The Barcelona Institute of Science and Technology, Baldiri Reixac, 10, 08028 Barcelona, Spain
Marina Gay
Institute for Research in Biomedicine (IRB Barcelona), The Barcelona Institute of Science and Technology, Baldiri Reixac, 10, 08028 Barcelona, Spain
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.
