Updated project metadata. 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 is another challenging question. 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, 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 pre-fractionation. This in-depth proteome analysis was accomplished by optimization of protein extraction, enzymatic digestion, LC gradient and peptide sequencing method. IPA analysis of protein exclusively identified in SCAD revealed several crucial signaling pathways which regulate breast cancer progression. An unbiased extraction of different categories of proteins associated with tumorigenesis was also demonstrated. This novel strategy not only expedites comprehensive protein identification but also post-translational modifications (PTMs) of proteins (e.g., glycosylation), thus is applicable for biomarker discovery in various types of cancers.