Abstract
BackgroundAn important step in the proteomics of solid tumors, including breast cancer, consists of efficiently extracting most of proteins in the tumor specimen. For this purpose, Radio-Immunoprecipitation Assay (RIPA) buffer is widely employed. RIPA buffer's rapid and highly efficient cell lysis and good solubilization of a wide range of proteins is further augmented by its compatibility with protease and phosphatase inhibitors, ability to minimize non-specific protein binding leading to a lower background in immunoprecipitation, and its suitability for protein quantitation.ResultsIn this work, the insoluble matter left after RIPA buffer extraction of proteins from breast tumors are subjected to another extraction step, using a urea-based buffer. It is shown that RIPA and urea lysis buffers fractionate breast tissue proteins primarily on the basis of molecular weights. The average molecular weight of proteins that dissolve exclusively in urea buffer is up to 60% higher than in RIPA.Gene Ontology (GO) and Directed Acyclic Graphs (DAG) are used to map the collective biological and biophysical attributes of the RIPA and urea proteomes. The Cellular Component and Molecular Function annotations reveal protein solubilization preferences of the buffers, especially the compartmentalization and functional distributions.It is shown that nearly all extracellular matrix proteins (ECM) in the breast tumors and matched normal tissues are found, nearly exclusively, in the urea fraction, while they are mostly insoluble in RIPA buffer. Additionally, it is demonstrated that cytoskeletal and extracellular region proteins are more soluble in urea than in RIPA, whereas for nuclear, cytoplasmic and mitochondrial proteins, RIPA buffer is preferred.Extracellular matrix proteins are highly implicated in cancer, including their proteinase-mediated degradation and remodelling, tumor development, progression, adhesion and metastasis. Thus, if they are not efficiently extracted by RIPA buffer, important information may be missed in cancer research.ConclusionFor proteomics of solid tumors, a two-step extraction process is recommended. First, proteins in the tumor specimen should be extracted with RIPA buffer. Second, the RIPA-insoluble material should be extracted with the urea-based buffer employed in this work.
Highlights
An important step in the proteomics of solid tumors, including breast cancer, consists of efficiently extracting most of proteins in the tumor specimen
It is shown that most extracellular matrix proteins (ECM) in the breast tumors and matched normal tissues are found, nearly exclusively, in the urea fraction, while they are mostly insoluble in RadioImmunoprecipitation Assay (RIPA) buffer
This work shows that most extracellular matrix proteins (ECM) in the breast tumors and matched normal tissues in this work are dissolved in the urea buffer fraction: they are mostly insoluble in RIPA buffer
Summary
An important step in the proteomics of solid tumors, including breast cancer, consists of efficiently extracting most of proteins in the tumor specimen For this purpose, RadioImmunoprecipitation Assay (RIPA) buffer is widely employed. An ideal sample prep protocol should isolate as much of the proteins of interest as possible from the biological source, and preserve optimal sample integrity and morphology It should present the entire sample in a form that is compatible with optimum mass spectrometric analysis. Proteins in their native states are generally embedded in their natural environments where they are associated with other proteins, biological macromolecules or other matrix materials. The bonds, and appropriate agents/methods for dissociating them [1] include: disulfide bond (reduction & alkylation), hydrogen bond (chaotropes), electrostatic interactions (salts, charged detergents, chaotropes), chargedipole (chaotropes), dipole-dipole (strong dipolar molecules), van der Waals (salt, dipolar molecules, chaotropes), and hydrophobic interactions (salts, dipolar molecules, chaotropes)
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