Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA.
Clin Proteomics. 2014 Feb 1;11(1):4. doi: 10.1186/1559-0275-11-4.
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.
在过去几十年中，已经制备了大量的福尔马林固定和石蜡包埋的临床组织，并将其储存在世界各地医院的病理储存库以及临床实验室中。除了存档组织外，为了灭活传染性物质，还必须从患病患者或动物模型中制备蛋白质组学样品，因此也需要福尔马林固定。由于蛋白质的氨基与甲醛之间形成席夫碱，因此从福尔马林固定的组织中提取蛋白质会受到阻碍。尽管从福尔马林固定的组织中获得蛋白质的最高提取效率对于获得最大的蛋白质组学信息至关重要，但尚未关注三羟甲基氨基甲烷（tris(hydroxymethyl)aminomethane）的浓度对提取效果的影响。我们怀疑三羟甲基氨基甲烷的浓度会影响蛋白质的提取效率，因为它是一种伯胺，可逆转蛋白质的伯胺与甲醛之间的席夫碱形成。因此，我们致力于优化蛋白质提取缓冲液的成分和方案，以实现从福尔马林固定和石蜡包埋组织中更好地提取蛋白质。
为了模拟从患病组织中提取蛋白质，我们用小鼠肝切片制备了福尔马林固定和石蜡包埋的样本，并通过在各种提取条件下改变蛋白质提取缓冲液成分三羟甲基氨基甲烷的浓度来研究提取效率和速度。正如预期的那样，我们发现三羟甲基氨基甲烷对从福尔马林固定和石蜡包埋的样本中提取蛋白质的性能（包括提取产量和提取速度）有显著影响。
我们建议在 90°C 下提取 90 分钟时，蛋白质提取缓冲液中的三羟甲基氨基甲烷浓度应高于 300 mM，以在更短的时间内实现最有效的蛋白质提取。该信息对于进行最有效的蛋白质提取至关重要，因为蛋白质组学分析需要从福尔马林固定和石蜡包埋的组织样本中提取蛋白质。
