Top-down identification and quantification of stable isotope labeled proteins from Aspergillus flavus using online nano-flow reversed-phase liquid chromatography coupled to a LTQ-FTICR mass spectrometer
Top-down identification and quantification of stable isotope labeled proteins from Aspergillus flavus using online nano-flow reversed-phase liquid chromatography coupled to a LTQ-FTICR mass spectrometer
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DOI：
10.1021/ac800254z
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发表时间：
2008-07-01
影响因子：
7.4
通讯作者：
Muddiman, David C.
中科院分区：
文献类型：
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作者：
Collier, Timothy S.;Hawkridge, Adam M.;Muddiman, David C.
完整蛋白质的在线液相色谱-质谱 (LC-MS) 分析（即自上而下的蛋白质组学）是质谱界不断发展的研究领域。自上而下的蛋白质 MS 表征的一个主要优点是，与更常见的自下而上的方法相比，完整蛋白质的信息得以保留，后者使用蛋白酶生成的肽来搜索基因组数据库以进行蛋白质鉴定。与蛋白质自上而下的 MS 表征同时出现的是细胞培养中氨基酸稳定同位素标记 (SILAC) 方法的开发和实施，用于通过 LC-MS 对蛋白质进行相对定量。在此，我们描述了使用直接耦合到线性离子阱傅里叶变换离子回旋共振质谱仪 (nLC-LTQ-FTICR-MS) 的纳流反相液相色谱对源自 SILAC 标记的黄曲霉的蛋白质进行定性和定量自上而下的表征。黄曲霉是一种有毒的丝状真菌，对农业经济和人类健康产生重大影响。 SILAC 标记提高了蛋白质鉴定的可信度，我们观察到了 1318 个独特的蛋白质质量，对应于 659 个 SILAC 对，其中 22 个被自信地鉴定。然而，我们发现使用自上而下的 MS/MS 分析 SILAC 标记蛋白质进行蛋白质定量时存在一些限制性问题。详细讨论了 SILAC 标记在竞争性内源产生的氨基酸残基存在下的作用及其对完整物种定量的影响。
Online liquid chromatography-mass spectrometric (LC-MS) analysis of intact proteins (i.e., top-down proteomics) is a growing area of research in the mass spectrometry community. A major advantage of top-down MS characterization of proteins is that the information of the intact protein is retained over the vastly more common bottom-up approach that uses protease-generated peptides to search genomic databases for protein identification. Concurrent to the emergence of top-down MS characterization of proteins has been the development and implementation of the stable isotope labeling of amino acids in cell culture (SILAC) method for relative quantification of proteins by LC-MS. Herein we describe the qualitative and quantitative top-down characterization of proteins derived from SILAC-labeled Aspergillus flavus using nanoflow reversed-phase liquid chromatography directly coupled to a linear ion trap Fourier transform ion cyclotron resonance mass spectrometer (nLC-LTQ-FTICR-MS). A. flavus is a toxic filamentous fungus that significantly impacts the agricultural economy and human health. SILAC labeling improved the confidence of protein identificaion, and we observed 1318 unique protein masses corresponding to 659 SILAC pairs, of which 22 were confidently identified. However, we have observed some limiting issues with regard to protein quantification using top-down MS/MS analyses of SILAC-labeled proteins. The role of SILAC labeling in the presence of competing endogenously produced amino acid residues and its impact on quantification of intact species are discussed in detail.