Use of differential isotopic labeling and mass spectrometry to analyze capacitation-associated changes in the phosphorylation status of mouse sperm proteins.
Use of differential isotopic labeling and mass spectrometry to analyze capacitation-associated changes in the phosphorylation status of mouse sperm proteins.
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DOI：
10.1021/pr800796j
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发表时间：
2009-03
影响因子：
4.4
通讯作者：
Visconti PE
中科院分区：
文献类型：
--
作者：
Platt MD;Salicioni AM;Hunt DF;Visconti PE
哺乳动物精子在获得生殖能力之前需要在雌性生殖道中停留一段有限的时间。与此过程相关的生化变化统称为“获能”。使用小鼠作为实验模型，我们以前已经证明，获能与cAMP依赖性蛋白酪氨酸磷酸化的增加。然而，很少有人知道这个磷酸化级联反应的蛋白质目标的身份和功能。在目前的工作中，我们已经使用了差分同位素标记结合固定化金属亲和色谱（IMAC）的磷酸肽富集和分析的混合线性离子阱/FT-ICR质谱仪来测量蛋白质磷酸化的获能过程中产生的变化。由于在精子样本的制备中未使用激酶激活剂和/或磷酸酶抑制剂，因此本研究中鉴定的磷酸化残基代表体内磷酸化位点。此外，与依赖于在蛋白质水平掺入同位素标记的氨基酸的其他方法（例如，SILAC），本技术基于蛋白质底物的Fisher酯化，允许在不存在蛋白质合成的情况下比较磷酸化状态。这种方法导致在55个独特的，在体内的磷酸化位点的鉴定和允许的相对程度的磷酸化，作为获能的结果，要计算42个不同的磷酸肽。这项工作代表了第一次努力，以确定特定的蛋白质磷酸化位点改变其磷酸化状态在体内作为哺乳动物获能过程的结果。
Mammalian sperm need to reside in the female reproductive tract for a finite period of time before acquiring fertilizing competence. The biochemical changes associated with this process are collectively known as “capacitation”. With the use of the mouse as an experimental model, we have previously demonstrated that capacitation is associated with a cAMP-dependent increase in protein tyrosine phosphorylation. However, little is known about the identity and function of the protein targets of this phosphorylation cascade. In the present work, we have used differential isotopic labeling coupled with immobilized metal affinity chromatography (IMAC)-based phosphopeptide enrichment and analysis on a hybrid linear ion trap/FT-ICR mass spectrometer to measure the changes in protein phosphorylation resulting from the capacitation process. As no kinase activators and/or phosphatase inhibitors were used in the preparation of the sperm samples, phosphorylated residues identified in this study represent in vivo sites of phosphorylation. Also, in contrast to other methods which rely on the incorporation of isotopically labeled amino acids at the protein level (e.g., SILAC), the present technique is based on the Fisher esterification of protein digests, allowing for the comparison of phosphorylation status in the absence of protein synthesis. This approach resulted in the identification of 55 unique, in vivo sites of phosphorylation and permitted the relative extent of phosphorylation, as a consequence of capacitation, to be calculated for 42 different phosphopeptides. This work represents the first effort to determine which specific protein phosphorylation sites change their phosphorylation status in vivo as a result of the mammalian capacitation process.
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