Efficient extraction of proteins from formalin-fixed paraffin-embedded tissues requires higher concentration of tris(hydroxymethyl)aminomethane.
Efficient extraction of proteins from formalin-fixed paraffin-embedded tissues requires higher concentration of tris(hydroxymethyl)aminomethane.
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DOI：
10.1186/1559-0275-11-4
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发表时间：
2014-02-01
影响因子：
3.8
通讯作者：
Matsumoto H
中科院分区：
文献类型：
--
作者：
Kawashima Y;Kodera Y;Singh A;Matsumoto M;Matsumoto H
在过去的几十年中，已经产生了许多福尔马林固定和石蜡包埋的临床组织，并储存在世界各地的医院以及临床实验室的病理学保藏中。除了存档的组织之外，甲醛脱固定也是制备来自患病患者或动物模型的蛋白质组学样品的强制性要求，以防止传染性病原体。从甲醛固定的组织中提取蛋白质受到蛋白质的氨基与甲醛之间形成席夫碱的阻碍。虽然从甲醛固定的组织中实现蛋白质的最高提取效率对于获得最大的蛋白质组学信息是必不可少的，但是没有注意到三（羟甲基）氨基甲烷对提取效率的浓度依赖性。我们怀疑三（羟甲基）氨基甲烷的浓度影响蛋白质提取效率，因为其作为伯胺的性质逆转蛋白质的伯胺与甲醛之间的席夫碱形成。因此，我们追求蛋白质提取缓冲液的组分和方案的优化，以实现从甲醛固定和石蜡包埋的组织中更好地提取蛋白质。为了模拟从病变组织中提取蛋白质，我们从小鼠肝切片中制备了甲醛固定和石蜡包埋的样品，并通过改变蛋白质提取缓冲液组分三（羟甲基）氨基甲烷的浓度来研究在各种提取条件下蛋白质提取效率和速度。我们发现，正如预期的那样，三（羟甲基）氨基甲烷显着影响从甲醛固定和石蜡包埋的样品中提取蛋白质的提取率和提取速度的性能。当在90°C下进行提取90 min时，我们建议蛋白质提取缓冲液中三（羟甲基）氨基甲烷的浓度高于300 mM，以在更短的时间内实现最有效的蛋白质提取。这些信息对于从甲醛固定和石蜡包埋的组织样品中进行最有效的蛋白质提取以进行蛋白质组学分析至关重要。
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. 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. 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.
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