Comparing multistep immobilized metal affinity chromatography and multistep TiO2 methods for phosphopeptide enrichment.
Comparing multistep immobilized metal affinity chromatography and multistep TiO2 methods for phosphopeptide enrichment.
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DOI：
10.1021/acs.analchem.5b01833
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发表时间：
2015-09-01
影响因子：
7.4
通讯作者：
Hummon AB
中科院分区：
文献类型：
--
作者：
Yue X;Schunter A;Hummon AB
从复杂的肽混合物中富集磷酸肽是基于质谱的磷酸蛋白质组学研究的重要步骤，以降低样品的复杂性和电离抑制效应。用于富集磷酸肽的典型方法包括固定化金属亲和层析（IMAC）或二氧化钛（TiO 2）珠，其具有选择性亲和力并且可以与磷酸肽相互作用。在这项研究中，IMAC富集方法与TiO 2富集方法进行了比较，使用多步富集策略，从全细胞裂解液，以评估其能力，以丰富不同类型的磷酸肽。针对IMAC和TiO 2珠优化肽与珠的比率。IMAC和TiO2富集均进行三轮，以能够从全细胞裂解物中最大限度地提取磷酸肽。分析了IMAC富集、TiO 2富集和IMAC和TiO 2富集鉴定的磷酸肽的特性。IMAC和TiO 2都富集了类似量的磷酸肽，具有可比的富集效率。然而，IMAC富集所特有的磷酸肽显示出更高百分比的多磷酸肽，以及更高百分比的更长、碱性和亲水性磷酸肽。此外，IMAC和TiO 2程序明显富集具有不同基序的磷酸肽。最后，在IMAC富集后，用两轮TiO 2从上清液进一步富集，或用两轮IMAC从上清液TiO 2富集进一步富集不能完全回收用相应的多步富集未鉴定的磷酸肽。
Phosphopeptide enrichment from complicated peptide mixtures is an essential step for mass spectrometry-based phosphoproteomic studies to reduce sample complexity and ionization suppression effects. Typical methods for enriching phosphopeptides include immobilized metal affinity chromatography (IMAC) or titanium dioxide (TiO2) beads, which have selective affinity and can interact with phosphopeptides. In this study, the IMAC enrichment method was compared with the TiO2 enrichment method, using a multi-step enrichment strategy from whole cell lysate, to evaluate their abilities to enrich for different types of phosphopeptides. The peptide-to-beads ratios were optimized for both IMAC and TiO2 beads. Both IMAC and TiO2 enrichments were performed for three rounds to enable the maximum extraction of phosphopeptides from the whole cell lysates. The phosphopeptides that are unique to IMAC enrichment, unique to TiO2 enrichment, and identified with both IMAC and TiO2 enrichment were analyzed for their characteristics. Both IMAC and TiO2 enriched similar amounts of phosphopeptides with comparable enrichment efficiency. However, phosphopeptides that are unique to IMAC enrichment showed a higher percentage of multi-phosphopeptides, as well as a higher percentage of longer, basic, and hydrophilic phosphopeptides. Also, the IMAC and TiO2 procedures clearly enriched phosphopeptides with different motifs. Finally, further enriching with two rounds of TiO2 from the supernatant after IMAC enrichment, or further enriching with two rounds of IMAC from the supernatant TiO2 enrichment does not fully recover the phosphopeptides that are not identified with the corresponding multi-step enrichment.