Systematic comparison between SDS-PAGE/RPLC and high-/low-pH RPLC coupled tandem mass spectrometry strategies in a whole proteome analysis.
Systematic comparison between SDS-PAGE/RPLC and high-/low-pH RPLC coupled tandem mass spectrometry strategies in a whole proteome analysis.
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DOI：
10.1039/c4an02119c
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发表时间：
2015-02
期刊：
影响因子：
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通讯作者：
Xuefei Yin;Yang Zhang;Xiaohui Liu;Cheng Chen;Haojie Lu;Huali Shen;Pengyuan Yang
中科院分区：
文献类型：
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作者：
Xuefei Yin;Yang Zhang;Xiaohui Liu;Cheng Chen;Haojie Lu;Huali Shen;Pengyuan Yang
SDS-PAGE和高ph RPLC是蛋白质组学研究中常用的分离方法。对这两种策略进行比较研究，有助于深入了解它们各自的特点。在此，我们通过4种SDS-PAGE/RPLC和3种高/低ph RPLC不同的工作流程对两种方法进行了系统的比较，以提高蛋白质鉴定的灵敏度。HepG2细胞共鉴定出9793个蛋白，其中高/低ph RPLC鉴定出8581个蛋白，SDS-PAGE/RPLC鉴定出7933个蛋白。结果表明，在第一维分离中使用高ph RPLC有利于高通量蛋白质组分析，但选择SDS-PAGE可以产生更好的肽覆盖。我们发现SDS-PAGE分离方法有利于中性pI肽。我们还分析了两种策略引起的意外变化。我们的研究结果表明，在这两种策略中，更多的预分离有利于蛋白质鉴定，并且根据凝胶片的灰色值进行池化可以提高SDS-PAGE/RPLC工作流程中的鉴定效率。
SDS-PAGE and high-pH RPLC are commonly used fractionation strategies in proteomics research. A comparative investigation of these two strategies would be meaningful to thoroughly understand their respective features. Here, we systematically compared the two methods by trying 4 SDS-PAGE/RPLC and 3 high-/low-pH RPLC different workflows for a higher sensitivity in protein identification. Totally 9793 proteins were identified in HepG2 cells, with 8581 proteins identified by high-/low-pH RPLC workflows and 7933 by SDS-PAGE/RPLC workflows. The results demonstrate that using high-pH RPLC in the first dimensional separation would favour a high-throughput proteome analysis but choosing SDS-PAGE could yield much better peptide coverage. We found that the SDS-PAGE fractionation method benefits the neutral pI peptides. We also analyzed unexpected modifications caused by the two strategies. Our results suggest that more pre-fractionation benefits protein identifications in both strategies and pooling of gel pieces according to their grey values increased the identification efficiency in SDS-PAGE/RPLC workflows.