Laboratoire de Spectrométrie de Masse des Protéines, Institut de Biologie Structurale, Grenoble, France.
J Am Soc Mass Spectrom. 2010 Jan;21(1):76-9. doi: 10.1016/j.jasms.2009.09.005. Epub 2009 Sep 17.
Structural studies of proteins by hydrogen/deuterium exchange coupled to mass spectrometry (DXMS) require the use of proteases working at acidic pH and low temperatures. The spatial resolution of this technique can be improved by combining several acidic proteases, each generating a set of different peptides. Three commercial aspartic proteases are used, namely, pepsin, and proteases XIII and XVIII. However, given their low purity, high enzyme/protein ratios have to be used with proteases XIII and XVIII. In the present work, we investigate the activity of two alternative acidic proteases from Plasmodium falciparum under different pH and temperature conditions. Peptide mapping of four different proteins after digestion with pepsin, plasmepsin 2 (PSM2), and plasmepsin 4 (PSM4) were compared. PSM4 is inactive at pH 2.2 and 0 degrees C, making it unusable for DXMS studies. However, PSM2 showed low but reproducible activity under DXMS conditions. It displayed no substrate specificity and, like pepsin, no strict sequence specificity. Altogether, these results show that PSM2 but not PSM4 is a potential new tool for DXMS studies.
通过与质谱联用的氢/氘交换研究蛋白质的结构（DXMS）需要使用在酸性 pH 和低温下工作的蛋白酶。通过组合几种酸性蛋白酶，每种蛋白酶产生一组不同的肽，可以提高该技术的空间分辨率。使用了三种商业天冬氨酸蛋白酶，即胃蛋白酶、蛋白酶 XIII 和 XVIII。然而，由于它们的纯度低，必须使用蛋白酶 XIII 和 XVIII 来实现高酶/蛋白比。在本工作中，我们在不同的 pH 和温度条件下研究了来自恶性疟原虫的两种替代酸性蛋白酶的活性。比较了用胃蛋白酶、疟原虫蛋白酶 2（PSM2）和疟原虫蛋白酶 4（PSM4）消化后四种不同蛋白质的肽图谱。PSM4 在 pH 2.2 和 0°C 下无活性，使其无法用于 DXMS 研究。然而，PSM2 在 DXMS 条件下表现出低但可重复的活性。它没有底物特异性，也不像胃蛋白酶那样具有严格的序列特异性。总之，这些结果表明 PSM2 而不是 PSM4 是 DXMS 研究的潜在新工具。
