Snijders Ambrosius P L, de Vos Marjon G J, de Koning Bart, Wright Phillip C
Biological and Environmental Systems Group, Department of Chemical and Process Engineering, University of Sheffield, Mappin Street, Sheffield S1 3JD, UK.
Electrophoresis. 2005 Aug;26(16):3191-9. doi: 10.1002/elps.200500218.
We provide a method for accurate protein quantitation that uses two-dimensional (2-D) gel electrophoresis for protein separation, but does not require extensive statistical analysis of staining intensities on gels. Instead, accurate quantitation occurs on the mass spectrometer (MAS) on multiple peptides to provide statistical evidence. In an example study, Sulfolobus solfataricus cells were grown on the carbon sources glucose, fructose and glutamate. The glucose phenotype (reference) was grown on (15)N-enriched medium. Next, the glutamate and the fructose phenotypes are mixed with the reference and two 2-D gels are created. Staining intensities of gel spots in this case are used for initial, semiquantitative assessment of differential expression. On this basis, spots are selected for accurate quantitation on the MAS. A number of differentially expressed proteins were found, for example: a (25.2 +/- 8.2)-fold upregulation of isocitrate lyase and a (7.14 +/- 0.82)-fold downregulation of glucose dehydrogenase on glutamate compared to glucose. With this protocol, intergel and interlaboratory comparisons are facilitated, since the light and heavy versions of a protein are equally affected by variations in sample preparation and buffer composition. Because the statistical evidence is gathered on the MAS, the need to run vast numbers of gels is removed.
我们提供了一种准确的蛋白质定量方法，该方法使用二维（2-D）凝胶电泳进行蛋白质分离，但不需要对凝胶上的染色强度进行广泛的统计分析。相反，通过对多个肽段在质谱仪（MAS）上进行准确的定量，以提供统计证据。在一项示例研究中，嗜热栖热菌细胞在碳源葡萄糖、果糖和谷氨酸上生长。葡萄糖表型（参照）在富含（15）N的培养基上生长。接下来，将谷氨酸和果糖表型与参照混合，并制作两块二维凝胶。在这种情况下，凝胶斑点的染色强度用于差异表达的初步半定量评估。在此基础上，选择斑点在质谱仪上进行准确的定量。发现了许多差异表达的蛋白质，例如：与葡萄糖相比，谷氨酸上异柠檬酸裂合酶上调了（25.2±8.2）倍，葡萄糖脱氢酶下调了（7.14±0.82）倍。采用该方案便于凝胶间和实验室间的比较，因为蛋白质的轻链和重链版本同样受到样品制备和缓冲液组成变化的影响。由于统计证据是在质谱仪上收集的，因此无需运行大量的凝胶。
