Efficient extraction of proteins from formalin-fixed paraffin-embedded tissues requires higher concentration of tris(hydroxymethyl)aminomethane

Clinical Proteomics

Masaomi Matsumoto³  Yusuke Kawashima⁴  Yoshio Kodera²  Anil Singh¹  Hiroyuki Matsumoto¹ 

[1] Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, USADepartment of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, USADepartment of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, USA;Laboratory of Biomolecular Dynamics, Department of Physics, School of Science, Kitasato University, Kanagawa, JapanCenter for Disease Proteomics, School of Science, Kitasato University, Kanagawa, JapanLaboratory of Biomolecular Dynamics, Department of Physics, School of Science, Kitasato University, Kanagawa, JapanLaboratory of Biomolecular Dynamics, Department of Physics, School of Science, Kitasato University, Kanagawa, JapanCenter for Disease Proteomics, School of Science, Kitasato University, Kanagawa, JapanCenter for Disease Proteomics, School of Science, Kitasato University, Kanagawa, JapanLaboratory of Biomolecular Dynamics, Department of Physics, School of Science, Kitasato University, Kanagawa, JapanCenter for Disease Proteomics, School of Science, Kitasato University, Kanagawa, Japan;Department of Chemistry, University of North Carolina, Chapel Hill, USADepartment of Chemistry, University of North Carolina, Chapel Hill, USADepartment of Chemistry, University of North Carolina, Chapel Hill, USA;Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, USALaboratory of Biomolecular Dynamics, Department of Physics, School of Science, Kitasato University, Kanagawa, JapanDepartment of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, USADepartment of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, USALaboratory of Biomolecular Dynamics, Department of Physics, School of Science, Kitasato University, Kanagawa, JapanLaboratory of Biomolecular Dynamics, Department of Physics, School of Science, Kitasato University, Kanagawa, JapanDepartment of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, USALaboratory of Biomolecular Dynamics, Department of Physics, School of Science, Kitasato University, Kanagawa, Japan

关键词: FFPE;    Protein extraction;    TRIS concentration;    Proteomics;   

DOI  :  10.1186/1559-0275-11-4

来源: Humana Press Inc

【 摘 要 】

Abstract

Background

Numerous formaldehyde-fixed and paraffin-embedded clinical tissues have been created in the past decades and stored in pathological depositories at hospitals as well as in clinical laboratories worldwide. In addition to the archived tissues, formaldehyde-fixation is also mandatory for preparing proteomics samples from diseased patients or animal models in order to inactivate contagious agents. Protein extraction from formaldehyde-fixed tissues is hampered by the Schiff base formation between the amino groups of proteins and formaldehyde. Although achievement of the highest extraction efficiency of proteins from the formaldehyde-fixed tissues is essential for obtaining maximum proteomics information, no attention has been paid to the concentration dependence of tris(hydroxymethyl)aminomethane on the extraction efficacy. We suspected that the concentration of tris(hydroxymethyl)aminomethane affects the protein extraction efficiency because of its property as a primary amine that reverses the Schiff base formation between the primary amines of proteins and formaldehyde. Thus we pursued optimization of the component and protocol of protein extraction buffer to achieve better extraction efficiency of proteins from formaldehyde-fixed and paraffin-embedded tissues.

Results

In order to simulate protein extraction from diseased tissues we made formaldehyde-fixed and paraffin-embedded samples from mouse liver slices and investigated the protein extraction efficiency and speed by changing the concentration of the protein extraction buffer component tris(hydroxymethyl)aminomethane under various extraction conditions. We find, as expected, that tris(hydroxymethyl)aminomethane significantly affects the performance of protein extraction from the formaldehyde-fixed and paraffin-embedded samples both in the extraction yield and in the extraction speed.

Conclusions

We recommend the concentration of tris(hydroxymethyl)aminomethane in protein extraction buffer to be higher than 300 mM when extraction is conducted for 90 min at 90°C to achieve the most efficient protein extraction in a shorter time. The information will be essential for performing the most efficient protein extraction from formaldehyde-fixed and paraffin-embedded tissue samples for proteomics analysis.

【 授权许可】

Unknown