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.