High-efficiency protein extraction from polyacrylamide gels for molecular mass measurement by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry
High-efficiency protein extraction from polyacrylamide gels for molecular mass measurement by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry
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DOI：
10.1002/elps.200410187
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发表时间：
2005-03-01
期刊：
影响因子：
2.9
通讯作者：
Manabe, T
中科院分区：
文献类型：
--
作者：
Jin, Y;Manabe, T
本文报道了一种简单、快速的从考马斯亮蓝（CBB）染色聚丙烯酰胺凝胶中提取蛋白质的方法，该方法适用于基质辅助激光解吸/电离飞行时间质谱（MALDITOF-MS）测定蛋白质的分子质量。在25℃下，0.1 m NaOH浸泡10 min，提取cbb染色凝胶片中的蛋白质，对于< 67 kDa的蛋白质，一步提取法的回收率为34-73%。在质谱分析过程中，通过碱提取前的染色步骤避免了CBB对蛋白质的内聚。在所有< 36kDa的蛋白中，提取蛋白的分子质量值与纯化蛋白的分子质量值在+/- 0.01 ~ 0.10%的偏差范围内吻合。由于提取率高，从cbb染色凝胶中提取的蛋白质可以进行质量测量，其中细胞色素c、α -乳清蛋白、肌红蛋白、β -乳糖球蛋白、胰蛋白酶原和碳酸酐酶（12.4-29.0kDa）的负载蛋白量低至34 ng，甘油醛-3-磷酸脱氢酶（35.6kDa）和白蛋白（66.3 kDa）的负载蛋白量为340ng。该方法为利用cbb染色的一维或二维凝胶进行MALDI-TOF-MS全蛋白分析提供了一种高效的方法。
A simple and fast method of protein extraction from Coomassie Brilliant Blue (CBB)stained polyacrylamide gels suited for molecular mass measurement of proteins by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDITOF-MS) is reported. Proteins in CBB-stained gel pieces were extracted by a 10-min soaking in 0.1 m NaOH at 25 degrees C. The recovery of this one-step extraction method was 34-73% for proteins < 67 kDa. CBB adduction to proteins during mass spectrometric analysis was avoided by a destaining step before the alkaline extraction. The molecular mass values of the extracted proteins coincided with those of purified proteins within +/- 0.01-0.10% deviation for all the proteins < 36kDa. Because of the high extraction recovery, mass measurement was possible for the proteins extracted from CBB-stained gels with loaded protein quantities as little as 34 ng for cytochrome c, alpha-lactalbumin, myoglobin, beta-actoglobulin, trypsinogen, and carbonic anhydrase (12.4-29.0kDa), 340ng for glyceraldehyde-3-phosphate dehydrogenase (35.6kDa) and albumin (66.3 kDa). This method provides a highly efficient approach to utilize CBB-stained one- or two-dimensional gels for whole protein analysis using MALDI-TOF-MS.