Extending the Mannose 6-Phosphate Glycoproteome by High Resolution/Accuracy Mass Spectrometry Analysis of Control and Acid Phosphatase 5-Deficient Mice
Extending the Mannose 6-Phosphate Glycoproteome by High Resolution/Accuracy Mass Spectrometry Analysis of Control and Acid Phosphatase 5-Deficient Mice
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DOI：
10.1074/mcp.m112.026179
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发表时间：
2013-07-01
影响因子：
7
通讯作者：
Lobel, Peter
中科院分区：
文献类型：
--
作者：
Sleat, David E.;Sun, Pengling;Lobel, Peter
在哺乳动物中，大多数新合成的管腔溶酶体蛋白是通过甘露糖6-磷酸(Man6P)靶向途径传递到溶酶体的。含Man6P的蛋白质可以用蛋白质组学方法进行亲和纯化和鉴定，这样的研究已经导致了新的溶酶体蛋白和相关的人类疾病基因的发现。这种方法的一个局限性是，在大多数细胞类型中，当蛋白质靶向溶酶体后，Man6P修饰会被酸性磷酸酶5(ACP5)迅速去除，因此，一些溶酶体蛋白可能逃脱检测。在这项研究中，我们扩展了使用高分辨率/准确质谱仪对溶酶体蛋白质组的分析，以在对照和ACP5缺陷小鼠的联合分析中鉴定和定量蛋白质。为了鉴定组织分布有限的Man6P糖蛋白，我们分析了多个组织，并使用统计方法来鉴定纯化的具有高特异性的蛋白质。除了68个已知的Man6P糖蛋白外，还鉴定了165个其他小鼠蛋白，它们可能含有Man6P，因此可能代表新的溶酶体居留。对于其中四个候选溶酶体(乳过氧化物酶、磷脂酶D家族成员3、核糖核酸酶6和血清淀粉样蛋白P组分)，我们证明了基于荧光融合蛋白与溶酶体标记共存的溶酶体居留。
In mammals, most newly synthesized lumenal lysosomal proteins are delivered to the lysosome by the mannose 6-phosphate (Man6P) targeting pathway. Man6P -containing proteins can be affinity-purified and characterized using proteomic approaches, and such studies have led to the discovery of new lysosomal proteins and associated human disease genes. One limitation to this approach is that in most cell types the Man6P modification is rapidly removed by acid phosphatase 5 (ACP5) after proteins are targeted to the lysosome, and thus, some lysosomal proteins may escape detection. In this study, we have extended the analysis of the lysosomal proteome using high resolution/accuracy mass spectrometry to identify and quantify proteins in a combined analysis of control and ACP5-deficient mice. To identify Man6P glycoproteins with limited tissue distribution, we analyzed multiple tissues and used statistical approaches to identify proteins that are purified with high specificity. In addition to 68 known Man6P glycoproteins, 165 other murine proteins were identified that may contain Man6P and may thus represent novel lysosomal residents. For four of these lysosomal candidates, (lactoperoxidase, phospholipase D family member 3, ribonuclease 6, and serum amyloid P component), we demonstrate lysosomal residence based on the colocalization of fluorescent fusion proteins with a lysosomal marker.