Surfactant and Chaotropic Agent Assisted Sequential Extraction/On-Pellet Digestion (SCAD) for Enhanced Proteomics

As a popular sample preparation approach, filter-aided sample preparation (FASP) has been widely used in proteomic analysis. However, several limitations have been noted, including sample loss during filtration, repetitive centrifugation steps, and the possibility of breakage of filtration membrane. Extraction bias among different sample preparation strategies presents another challenge. To overcome these limitations and address remaining challenges, we developed a novel surfactant and chaotropic agent assisted sequential extraction/on-pellet digestion (SCAD) protocol. The new strategy resulted in higher protein yield and improved peptide recovery and protein coverage compared to two conventional sample preparation methods (FASP and urea). In combination of three strategies, more than 10,000 distinct protein groups were identified with 1% FDR from MDA-MB-231 cells without any prefractionation. This in-depth proteome analysis was accomplished by optimization of protein extraction, enzymatic digestion, LC gradient, and peptide sequencing method. Ingenuity Pathways Analysis (IPA) of proteins exclusively identified in SCAD revealed several crucial signaling pathways that regulate breast cancer progression. SCAD also enabled an unbiased extraction of different categories of proteins (membrane, intracellular, nuclear) associated with tumorigenesis, which integrates the advantages of FASP and urea extraction. This novel strategy expedites comprehensive protein identification, which is applicable for biomarker discovery in various types of cancers.作为一种主流的样品制备手段，滤膜辅助样品制备（filter-aided sample preparation, FASP）已被广泛应用于蛋白质组学分析领域。然而该方法存在诸多局限：包括过滤过程中的样品损失、重复离心操作，以及滤膜破损的风险；不同样品制备策略间存在的提取偏倚，亦构成了另一项研究挑战。为克服上述局限并解决现存挑战，本研究开发了一种全新的表面活性剂与离液剂辅助连续提取/颗粒内消化（surfactant and chaotropic agent assisted sequential extraction/on-pellet digestion, SCAD）流程。相较于两种传统样品制备方法（FASP与尿素提取法），该新策略可获得更高的蛋白质得率，同时提升肽段回收率与蛋白质覆盖度。将三种策略联合使用时，无需任何预分级步骤，即可从MDA-MB-231细胞中以1%的假发现率（false discovery rate, FDR）鉴定出超过10000个独立蛋白质组。本研究通过优化蛋白质提取、酶解、液相色谱（liquid chromatography, LC）梯度以及肽段测序方法，完成了此次深度蛋白质组学分析。对仅由SCAD策略特异性鉴定得到的蛋白质进行IPA通路分析（Ingenuity Pathways Analysis, IPA），揭示了多条调控乳腺癌进展的关键信号通路。SCAD策略还可实现与肿瘤发生相关的不同类别蛋白质（膜蛋白、胞内蛋白、核蛋白）的无偏倚提取，同时整合了FASP与尿素提取法的优势。这一全新策略可加速全面的蛋白质鉴定工作，适用于多种癌症的生物标志物发现研究。