The protamine 1 (P1) and protamine 2 (P2) family comprises the most abundant basic proteins in human spermatozoa and packs 85-95% of the paternal genome. P1 is synthesized as a mature form, whereas P2 components (HP2, HP3, and HP4) are generated from the proteolysis of the precursor. The particular physical-chemical properties of protamines hinder their identification by the standard bottom-up mass spectrometry (MS) strategy. A top-down MS approach is therefore proposed to identify human protamines and their post-translational modifications (PTMs) at the molecular level.
The intact protamine-enriched fraction from a single man was isolated from 15 million purified spermatozoa after histone removal and disulfide bonds reduction. Protamine enrichment was verified through acid-urea PAGE. The protamine-enriched fraction was analyzed by nanoLC-MS/MS using an Orbitrap Fusion Lumos mass spectrometer. Data analysis was performed using Proteome Discoverer 2.1 with Prosight PD 4.0 and Sequest HT nodes and TopPIC software.
The top-down proteomic MS approach mainly allowed the identification of the intact naïve P1, while HP2 and HP3 were detected with minor peak intensities. A phosphorylation pattern in P1 and combinations of other PTMs among the different protamines were detected. Notably, a hyperoxidation on cysteine residues modified to sulfinic acid was found in almost 100% of HP2 and HP3 and in nearby 70% of HP1, most likely generated in vivo, because of the presence of reactive oxygen species (ROS). However, the mass of the oxidized HP2 totally matches with an alternative spliced variant of P2 identified at RNA level by our group and others. Incorporation of middle-down MS strategies would allow differentiating between the potential alternative spliced variant of P2 and the hyperoxidized HP2.
The top-down MS approach allows the characterization of protamine PTM profiles at the intact level The establishment of the normal protamine PTMs profile in fertile individuals and the identification of pattern alterations in different types of infertile patients, including those with abnormal elevated ROS, would provide insights into the role of protamine PTMs code to male fertility and its potential function as epigenetic mark during early stages of preimplantation embryogenesis.