Department of Chemical Physiology, The Scripps Research Institute, 10550 N. Torrey Pines Rd., La Jolla, California 92037, USA.
J Proteome Res. 2012 May 4;11(5):2697-709. doi: 10.1021/pr300200x. Epub 2012 Apr 17.
Herein we report the characterization and optimization of single-step inline enrichment of phosphopeptides directly from small amounts of whole cell and tissue lysates (100-500 μg) using a hydroxyapatite (HAP) microcolumn and Multidimensional Protein Identification Technology (MudPIT). In comparison to a triplicate HILIC-IMAC phosphopeptide enrichment study, ∼80% of the phosphopeptides identified using HAP-MudPIT were unique. Similarly, analysis of the consensus phosphorylation motifs between the two enrichment methods illustrates the complementarity of calcium- and iron-based enrichment methods and the higher sensitivity and selectivity of HAP-MudPIT for acidic motifs. We demonstrate how the identification of more multiply phosphorylated peptides from HAP-MudPIT can be used to quantify phosphorylation cooperativity. Through optimization of HAP-MudPIT on a whole cell lysate we routinely achieved identification and quantification of ca. 1000 phosphopeptides from a ∼1 h enrichment and 12 h MudPIT analysis on small quantities of material. Finally, we applied this optimized method to identify phosphorylation sites from a mass-limited mouse brain region, the amygdala (200-500 μg), identifying up to 4000 phosphopeptides per run.
在此，我们报告了使用羟基磷灰石（HAP）微柱和多维蛋白质鉴定技术（MudPIT）直接从小量全细胞和组织裂解物（100-500μg）中一步在线富集磷酸肽的特征和优化。与三倍重复 HILIC-IMAC 磷酸肽富集研究相比，使用 HAP-MudPIT 鉴定的磷酸肽中约有 80%是独特的。同样，对两种富集方法之间的共识磷酸化基序的分析说明了钙基和铁基富集方法的互补性，以及 HAP-MudPIT 对酸性基序的更高灵敏度和选择性。我们展示了如何从 HAP-MudPIT 中鉴定更多的多磷酸化肽来定量磷酸化协同作用。通过对全细胞裂解物的 HAP-MudPIT 进行优化，我们通常可以从小量材料的约 1 小时富集和 12 小时 MudPIT 分析中鉴定和定量约 1000 个磷酸肽。最后，我们将这种优化方法应用于从质量有限的小鼠大脑区域（杏仁核，200-500μg）中鉴定磷酸化位点，每个运行可鉴定多达 4000 个磷酸肽。
