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首页> 外文期刊>Journal of proteome research >Characterization of Human Sperm Protamine Proteoforms through a Combination of Top-Down and Bottom-Up Mass Spectrometry Approaches
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Characterization of Human Sperm Protamine Proteoforms through a Combination of Top-Down and Bottom-Up Mass Spectrometry Approaches

机译：通过自上而下和自下而上的大众光谱法的组合表征人精子预蛋白蛋白族

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摘要

Protamine 1 (P1) and protamine 2 (P2) family are extremely basic, sperm-specific proteins, packing 85-95% of the paternal DNA. P1 is synthesized as a mature form, whereas P2 components (HP2, HP3, and HP4) arise from the proteolysis of the precursor (pre-P2). Due to the particular protamine physical-chemical properties, their identification by standardized bottom-up mass spectrometry (MS) strategies is not straightforward. Therefore, the aim of this study was to identify the sperm protamine proteoforms profile, including their post-translational modifications, in normozoospermic individuals using two complementary strategies, a top-down MS approach and a proteinase-K-digestion-based bottom-up MS approach. By top-down MS, described and novel truncated P1 and pre-P2 proteoforms were identified. Intact P1, pre-P2, and P2 mature proteoforms and their phosphorylation pattern were also detected. Additionally, a +61 Da modification in different proteoforms was observed. By the bottom-up MS approach, phosphorylated residues for pre-P2, as well as the new P2 isoform 2, which is not annotated in the UniProtKB database, were revealed. Implementing these strategies in comparative studies of different infertile phenotypes, together with the evaluation of P1/P2 and pre-P2/P2 MS-derived ratios, would permit determining specific alterations in the protamine proteoforms and elucidate the role of phosphorylation/dephosphorylation dynamics in male fertility.
机译：protamine1（p1）和protamine 2（p2）家族是极其基本的精子特异性蛋白质，包装85-95％的父DNA。 P1被合成为成熟形式，而P2组分（HP2，HP3和HP4）出现来自前体的蛋白水解（Pre-P2）。由于特定的protamine物理化学性质，它们通过标准化自下而上质谱（MS）策略的鉴定并不简单。因此，本研究的目的是鉴定使用两种互补策略，自上而下的MS方法和基于蛋白酶-K-消化的自下而上MS的常生症体中的精子protamine蛋白质曲线概况，包括其翻译后修饰，包括其翻译后修饰。方法。通过自上而下的MS，鉴定了所描述的和新的截短的P1和P2预蛋白染料。还检测到完整的P1，P2和P2成熟蛋白质常规及其磷酸化模式。另外，观察到不同蛋白质常规的+61DaDa改性。通过自下而上的MS方法，揭示了P2的磷酸化残基，以及在Uniprotkb数据库中未注释的新的P2同种型2。在不同不育表型的比较研究中实施这些策略，以及P1 / P2和P2 / P2衍生的比率的评价，将允许确定Protamine蛋白质常规中的特异性改变，并阐明磷酸化/去磷酸化动力学在男性中的作用生育能力。

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