Abstract
BackgroundNumerous 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.ResultsIn 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.ConclusionsWe 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.
Highlights
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
For Formaldehyde fixation and paraffin embedding (FFPE) tissues we used liver tissues of male C57BL/6 mice that were fixed by 10% formaldehyde and embedded in paraffin
The protein extraction efficiency was evaluated by band intensities of 78 kDa glucose-regulated protein (Grp78), argininosuccinate synthase (Ass) and lactate dehydrogenase A (Ldha) observed in Western blotting (Figure 1)
Summary
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. Protein extraction from formaldehyde-fixed tissues is hampered by the Schiff base formation between the amino groups of proteins and formaldehyde. 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. Large repositories of healthy as well as pathological FFPE tissues have been generated and stored worldwide. These FFPE samples are associated with clinical information concerning molecules in the tissue through Schiff base formation [3], hampering molecular investigation of these samples. For an effective proteomics analysis of necropsy or biopsy specimens, effective protein extraction from FFPE tissues is an absolute requirement [6]
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