Tip-Based Fractionation of Batch-Enriched Phosphopeptides Facilitates Easy and Robust Phosphoproteome Analysis,Journal of Proteome Research

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Tip-Based Fractionation of Batch-Enriched Phosphopeptides Facilitates Easy and Robust Phosphoproteome Analysis
Journal of Proteome Research ( IF 3.8 ) Pub Date : 2017-11-10 00:00:00 , DOI: 10.1021/acs.jproteome.7b00256
Alireza Dehghani ₁ , Markus Gödderz ₁ , Dominic Winter ₁

Affiliation

The identification of large numbers of phosphopeptides from complex samples largely relies on sample fractionation to reduce complexity and allow using large amounts of starting material. For such experiments, commonly fractionation of whole cell lysate digests followed by enrichment of phosphopeptides from the single fractions is performed. We evaluated the tip-based fractionation of batch-enriched phosphopeptides as an alternative method. We compared three tip-based fractionation methods employing strong cation exchange (SCX), strong anion exchange (SAX), and C18 material for basic reversed-phase (BRP) fractionation using HeLa whole cell lysate digests. We show that SCX tips are superior to BRP and SAX tips due to a more efficient retention and distribution of phosphopeptides as well as a better resolution. Furthermore, we show that tip-based fractionation results in a similar performance as fractionation followed by phosphopeptide enrichment of the single fractions and outperforms analysis of unfractionated phosphopeptide-enriched samples with long chromatography gradients. Our fractionation approach using SCX tips is straightforward, reproducible, and requires a fraction of time, effort, and instrumentation compared to those of the fractionation of whole cell lysate digests with subsequent enrichment of phosphopeptides from the single fractions.

中文翻译：

批次富集的磷酸肽的基于提示的分级分离有助于简便而稳健的磷酸化蛋白质组学分析

从复杂样品中鉴定大量磷酸肽很大程度上依赖于样品分级分离，以降低复杂性并允许使用大量的起始原料。对于这样的实验，通常进行全细胞裂解物消化物的分级分离，然后从单个级分中富集磷酸肽。我们评估了基于尖端的分批富集磷酸肽的分级分离方法。我们比较了使用强阳离子交换（SCX），强阴离子交换（SAX）和C18材料的三种基于尖端的分馏方法，这些方法用于使用HeLa全细胞裂解物消化液进行基本反相（BRP）分离。我们显示，由于更有效的磷酸肽保留和分布以及更好的分离度，SCX吸头优于BRP和SAX吸头。此外，我们显示，基于尖端的分级分离所得到的性能与分级分离后的性能相似，随后是单个级分的磷酸肽富集，并且具有长色谱梯度的未分级分离的富含磷肽的样品的性能优于常规分析。与全细胞裂解液消化物的分级分离以及随后从单个馏分中富集的磷酸肽相比，我们使用SCX尖端进行分级分离的方法是简单，可重现的，并且需要花费大量的时间，精力和仪器。

更新日期：2017-11-11

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