Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada.
Anal Chem. 2012 Jul 17;84(14):6181-91. doi: 10.1021/ac301169q. Epub 2012 Jul 6.
Protein solubilization is a key step in mass spectrometry-based shotgun proteome analysis. We describe a microwave-assisted protein solubilization (MAPS) method to dissolve proteins in reagents, such as NH(4)HCO(3) and urea, with high efficiency and with an added benefit that the solubilized proteins are denatured to become more susceptible to trypsin digestion, compared to other conventional protein solubilization techniques. In this method, a sample vial containing proteins suspended in a solubilization reagent is placed inside a domestic microwave oven and subjected to microwave irradiation for 30 s, followed by cooling the sample on ice to room temperature (~40 s) and then intermittent homogenization by vortex for 2 min. This cycle of microwave irradiation, cooling, and homogenization is repeated six times. In this way, sample overheating can be avoided, and a maximum amount of protein can be dissolved. It was shown that in the case of trypsin digestion of bovine serum albumen (BSA) more peptides and higher sequence coverage could be obtained from the protein dissolved by the MAPS method than the conventional heating, sonication, or vortex method. Compared to the most commonly used vortex-assisted protein solubilization method, MAPS reduces the solubilization time significantly, increases the amount of protein dissolvable in a reagent, and increases the number of proteins and peptides identified from a proteome sample. For example, in the proteome analysis of an Escherichia coli K-12 integral membrane protein extract, the MAPS method in combination with sequential protein solubilization and shotgun two-dimensional liquid chromatography tandem mass spectrometry analysis identified a total of 1291 distinct proteins and 10363 peptides, compared to 1057 proteins and 6261 peptides identified using the vortex method. Because MAPS can be done using an inexpensive microwave oven, this method can be readily adopted.
蛋白质溶解是基于质谱的鸟枪法蛋白质组分析的关键步骤。我们描述了一种微波辅助蛋白质溶解（MAPS）方法，该方法可以高效地将蛋白质溶解在试剂中，如 NH4HCO3和尿素，并且与其他常规蛋白质溶解技术相比，溶解的蛋白质变性，更易于胰蛋白酶消化。在该方法中，将含有悬浮在溶解试剂中的蛋白质的样品小瓶置于家用微波炉中，并进行微波辐射 30 秒，然后将样品冷却至冰上的室温（约 40 秒），然后通过涡旋间歇均质化 2 分钟。该微波辐射、冷却和均化循环重复六次。通过这种方式，可以避免样品过热，并可以溶解最大量的蛋白质。结果表明，在用胰蛋白酶消化牛血清白蛋白（BSA）时，通过 MAPS 方法溶解的蛋白质可以获得更多的肽段和更高的序列覆盖率，而通过常规加热、超声或涡旋方法则无法获得。与最常用的涡旋辅助蛋白质溶解方法相比，MAPS 显著缩短了溶解时间，增加了试剂中可溶解的蛋白质量，并增加了从蛋白质组样品中鉴定出的蛋白质和肽段的数量。例如，在大肠杆菌 K-12 整膜蛋白提取物的蛋白质组分析中，MAPS 方法与顺序蛋白质溶解和鸟枪法二维液相色谱串联质谱分析相结合，共鉴定出 1291 种独特的蛋白质和 10363 种肽段，而使用涡旋方法仅鉴定出 1057 种蛋白质和 6261 种肽段。由于 MAPS 可以使用廉价的微波炉进行，因此这种方法可以很容易地被采用。
