Nilsson C L, Puchades M, Westman A, Blennow K, Davidsson P
Department of Clinical Neuroscience, Göteborg University, Sahlgrenska University Hospital/Mölndal, Sweden.
Electrophoresis. 1999 Apr-May;20(4-5):860-5. doi: 10.1002/(SICI)1522-2683(19990101)20:4/5<860::AID-ELPS860>3.0.CO;2-I.
Pleural effusion may occur in patients suffering from physical trauma or systemic disorders such as infection, inflammation, or cancer. In order to investigate proteins in a pleural exudate from a patient with severe pneumonia, we used a strategy that combined preparative two-dimensional liquid-phase electrophoresis (2-D LPE), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and Western blotting. Preparative 2-D LPE is based on the same principles as analytical 2-D gel electrophoresis, except that the proteins remain in liquid phase during the entire procedure. In the first dimension, liquid-phase isoelectric focusing allows for the enrichment of proteins in liquid fractions. In the Rotofor cell, large volumes (up to 55 mL) and protein amounts (up to 1-2 g) can be loaded. Several low abundance proteins, cystatin C, haptoglobin, transthyretin, beta2-microglobulin, and transferrin, were detected after liquid-phase isoelectric focusing, through Western blotting analysis, in a pleural exudate (by definition, >25 g/L total protein). Direct MALDI-TOF-MS analysis of proteins in a Rotofor fraction is demonstrated as well. MALDI-TOF-MS analysis of a tryptic digest of a continuous elution sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) fraction confirmed the presence of cystatin C. By applying 2-D LPE, MALDI-TOF-MS, and Western blotting to the analysis of this pleural exudate, we were able to confirm the identity of proteins of potential diagnostic value. Our findings serve to illustrate the usefulness of this combination of methods in the analysis of pathological fluids.
胸腔积液可能发生在遭受身体创伤或患有全身性疾病（如感染、炎症或癌症）的患者身上。为了研究一名重症肺炎患者胸腔渗出液中的蛋白质，我们采用了一种将制备型二维液相电泳（2-D LPE）、基质辅助激光解吸/电离飞行时间质谱（MALDI-TOF-MS）和蛋白质印迹法相结合的策略。制备型2-D LPE基于与分析型二维凝胶电泳相同的原理，不同之处在于蛋白质在整个过程中保持液相状态。在第一维中，液相等电聚焦可使蛋白质在液体组分中得到富集。在Rotofor细胞中，可以加载大量（高达55 mL）的样品和蛋白质（高达1-2 g）。通过蛋白质印迹分析，在胸腔渗出液（根据定义，总蛋白>25 g/L）中液相等电聚焦后检测到了几种低丰度蛋白质，如胱抑素C、触珠蛋白、转甲状腺素蛋白、β2-微球蛋白和转铁蛋白。还展示了对Rotofor组分中的蛋白质进行直接MALDI-TOF-MS分析。对连续洗脱的十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）组分的胰蛋白酶消化产物进行MALDI-TOF-MS分析，证实了胱抑素C的存在。通过将2-D LPE、MALDI-TOF-MS和蛋白质印迹法应用于该胸腔渗出液的分析，我们能够确认具有潜在诊断价值的蛋白质的身份。我们的研究结果说明了这种方法组合在病理液体分析中的实用性。
