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.
Keywords: FASP; LFQ; Protein extraction; SCAD; breast cancer; enzymatic digestion; iBAQ; peptide sequencing; protein identification; urea.