Multidimensional protein identification (MudPIT), developed in the Yates Laboratory 20 years ago, is regarded as a powerful tool for proteomics research. Due to its remarkable online separation advantages, MudPIT has been widely used to facilitate discoveries in the field of proteomics research. However, it has one major disadvantage: the process of eluting peptides during strong cation exchange introduces salts, of different concentrations, into the mass spectrometer. Considering the sensitivity of the new generation of high-resolution mass spectrometers, developing a new version of MudPIT that could eliminate the introduction of salts in the elute would be a significant advancement to current technology. Herein, we developed a new, clean version of MudPIT called parallel channels-multidimensional protein identification technology (PC-MudPIT) to overcome this issue. In this design, the original biphasic trapping column was replaced by two parallel analytical column channels. We successfully averted the salt contamination yet retained all the other advantages of MudPIT. A total of 8161 and 7359 protein groups were identified from A549 whole cell lysate using PC-MudPIT and classic MudPIT, respectively. Moreover, we discovered the additional advantage that, in online mode, PC-MudPIT can also be used for an enrichment process of phosphopeptide identification. We identified a total 11453 phosphopeptides using PC-MudPIT and 7729 phosphopeptides using offline TiO2 enrichment followed by classic MudPIT. These advances indicate the possibility of other innovative applications of PC-MudPIT technology in deep proteome exploration.

中文翻译：

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并行通道多维蛋白质鉴定技术。

20年前在耶茨实验室开发的多维蛋白质鉴定（MudPIT）被认为是蛋白质组学研究的有力工具。由于其显着的在线分离优势，MudPIT已被广泛用于促进蛋白质组学研究领域的发现。但是，它有一个主要缺点：在强阳离子交换过程中洗脱肽的过程会将不同浓度的盐引入质谱仪中。考虑到新一代高分辨率质谱仪的灵敏度，开发可消除洗脱液中盐分引入的新版MudPIT将是当前技术的重大进步。在这里，我们开发了一种新的 干净的MudPIT版本称为并行通道多维蛋白质识别技术（PC-MudPIT），可以解决此问题。在此设计中，原来的双相捕集柱被两个平行的分析柱通道所取代。我们成功地避免了盐污染，但保留了MudPIT的所有其他优点。使用PC-MudPIT和经典MudPIT从A549全细胞裂解物中分别鉴定出总共8161和7359个蛋白质组。此外，我们发现了在线模式下PC-MudPIT还可以用于磷酸肽鉴定的富集过程的附加优势。我们使用PC-MudPIT识别了总共11453个磷酸肽，使用脱机TiO2富集然后进行了经典MudPIT识别了7729个磷酸肽。