An Optimized Platform for Hydrophilic Interaction Chromatography–Immobilized Metal Affinity Chromatography Enables Deep Coverage of the Rat Liver Phosphoproteome

While analysis of the phosphoproteome has become an important component of understanding how cells function, it remains a nontrivial task in terms of the number of sample preparation steps and instrument time needed to achieve sufficient depth of coverage to produce meaningful results. We previously described a multidimensional method that uses hydrophilic interaction chromatography (HILIC) followed by Fe3+ immobilized metal affinity chromatography (IMAC) to reduce complexity, improve selectivity, and increase phosphopeptide identifications. Here we present refinements to our overall protocol that make it simpler and more efficient, while they provide greater coverage of the phosphoproteome. We introduce filter-aided sample prep (FASP) for cell lysis and trypsin digestion. Following HILIC separation, fractions are IMAC enriched using a 96-well filter plate. Finally, enriched samples are analyzed using an LC–MS strategy optimized for the fractionation scheme. The optimized protocol improves protein recovery, simplifies phosphopeptide enrichment, and optimizes instrument time, while it maintains deep coverage of the phosphoproteome. By using the refined protocol, we identified more than 16,000 unique phosphosites from rat liver in a single experiment, which used approximately 1 day of instrument time. All together, we present evidence for 24,485 rat liver phosphosites that represents the deepest coverage of a tissue phosphoproteome to date.尽管磷酸化蛋白质组（phosphoproteome）分析已成为解析细胞功能机制的重要组成部分，但要获得足够深的覆盖度以产出有意义的研究结果，其所需的样品制备步骤与仪器耗时仍是一项颇具挑战的工作。此前我们曾报道一种多维分析方法，采用亲水相互作用色谱（hydrophilic interaction chromatography, HILIC）结合三价铁固定化金属亲和色谱（Fe3+ immobilized metal affinity chromatography, IMAC）以降低样品复杂度、提升分离选择性并增加磷酸肽的鉴定数目。本文中，我们对原有实验流程进行了优化改良，使其更趋简便高效，同时实现了更广范围的磷酸化蛋白质组覆盖。我们引入辅助过滤样品制备法（filter-aided sample prep, FASP）用于细胞裂解与胰蛋白酶消化。经HILIC分离后，我们采用96孔过滤板完成IMAC富集步骤。最后，适配该分级分离方案的液相色谱-质谱联用（LC–MS）分析策略被用于富集后的样品检测。经优化后的实验流程提升了蛋白质回收率，简化了磷酸肽富集流程，优化了仪器使用时长，同时仍可维持磷酸化蛋白质组的深度覆盖效果。通过使用该优化后的实验流程，我们在单次实验中仅耗费约1天的仪器时长，便从大鼠肝脏组织中鉴定出逾16000个独特磷酸化位点（phosphosites）。综上，本研究共鉴定得到24485个大鼠肝脏磷酸化位点，代表了当前已报道的组织磷酸化蛋白质组的最高覆盖深度。