Utility of electrophoretically derived protein mass estimates as additional constraints in proteome analysis of human serum based on MS/MS analysis
Utility of electrophoretically derived protein mass estimates as additional constraints in proteome analysis of human serum based on MS/MS analysis
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DOI：
10.1002/pmic.200401220
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发表时间：
2005-08-01
期刊：
影响因子：
3.4
通讯作者：
Yoo, JS
中科院分区：
文献类型：
--
作者：
Kim, JY;Lee, JH;Yoo, JS
使用多维分离技术在蛋白质和肽水平上分析HUPO人血清参考样品的蛋白质组。为了消除假阳性鉴定的搜索结果，我们采用了数据过滤方法，使用分子量（MW）的相关性来自变性1-DE。首先，使用免疫亲和层析从样品中除去六种最丰富的血清蛋白。根据分子量大小，用1-DE对剩余的血清蛋白进行分离，用胰蛋白酶进行胶内消化，用2-D LC/ESI-MS/MS对所得肽段进行分析。使用MS/MS结果进行SEQUEST搜索，鉴定出494个蛋白。其中，使用蛋白质数据过滤正式排除了202个，因为它们是单一分配蛋白质，并且它们的理论和质谱推导的MW在高置信度下不相关。为了评价该方法，将其结果与1-D LC/MALDI-TOF/TOF和HUPO血浆蛋白质组计划分析的结果进行比较。我们的数据过滤方法在分析复杂的大规模蛋白质组（如人血清）方面证明是有价值的。
The proteome of a HUPO human serum reference sample was analyzed using multidimensional separation techniques at both the protein and the peptide levels. To eliminate false-positive identifications from the search results, we employed a data filtering method using molecular weight (MW) correlations derived from denaturing 1-DE. First, the six most abundant serum proteins were removed from the sample using immunoaffinity chromatography. 1-DE was then used to fractionate the remaining serum proteins according to the MW Gel bands were isolated and in-gel digested with trypsin, and the resulting peptides were analyzed by 2-D LC/ESI-MS/MS. A SEQUEST search using the MS/MS results identified 494 proteins. Of these, 202 were excluded formally using protein data filtering as they were single-assignment proteins and their theoretical and electrophoretically-derived MWs did not correlate at high confidence. To evaluate this method, the results were compared with those of 1-D LC/MALDI-TOF/TOF and HUPO Plasma Proteome Project analyses. Our data filtering approach proved valuable in analysis of complex, large-scale proteomes such as human serum.