The identification of large numbers of phosphopeptides from complex samples largely relies on sample fractionation in order to reduce complexity and to 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 the fractionation of whole cell lysate digests with subsequent enrichment of phosphopeptides from the single fractions.

中文翻译：

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基于尖端的分批富集磷酸肽的分级分离有助于简便而稳健的磷酸化蛋白质组分析。

从复杂样品中鉴定大量磷酸肽很大程度上取决于样品的分级分离，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物质的三种基于尖端的分馏方法，用于使用HeLa全细胞裂解物消化液进行基本反相（BRP）分馏。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，基于尖端的分馏结果与分馏结果相似，随后是单个馏分的磷酸肽富集，并且在长色谱梯度下对未分离的磷酸肽富集的样品的性能优于分析。与对全细胞裂解液消化物进行分级分离以及随后从单个级分中富集磷酸肽相比，我们使用SCX尖端进行分级分离的方法简单，可重现，并且需要花费时间，精力和仪器。