Wang Haixing, Kachman Maureen T, Schwartz Donald R, Cho Kathleen R, Lubman David M
Department of Chemistry, School of Medicine, The University of Michigan, Ann Arbor, MI 48109, USA.
Electrophoresis. 2002 Sep;23(18):3168-81. doi: 10.1002/1522-2683(200209)23:18<3168::AID-ELPS3168>3.0.CO;2-A.
A molecular weight map of the protein content of ES2 human clear cell ovarian carcinoma cells has been produced using a two-dimensional (2-D) liquid separations/mass mapping technique. This method uses a 2-D liquid separation of proteins from whole cell lysates coupled on-line to an electrospray ionization-time of flight (ESI-TOF) mass spectrometer to map the accurate intact molecular weight (M(r)) of the protein content of the cells. The two separation dimensions involve the use of liquid isoelectric focusing as the first phase and nonporous silica reversed-phase high-performance liquid chromatography (HPLC) as the second phase of separation. The detection by ESI-TOF-MS provides an image of pI versus M(r) analogous to 2-D gel electrophoresis. Each protein is then identified based upon matrix-assisted laser desorption/ionization (MALDI)-TOF-MS peptide mapping and intact M(r) so that a standard map is produced against which other ovarian carcinoma cell lines can be compared. The accurate intact M(r) together with the pI fraction, and peptide map serve to tag the protein for future interlysate comparisons. An internal standard is also used to provide a means for quantitation for future interlysate studies. In the ES2 cell line under study it is shown that nearly 900 M(r) bands are detected over 17 pI fractions from pH 4 to 12 and a M(r) range up to 85 kDa and that around 290 of these bands can be identified using mass spectrometric based techniques. The protein M(r) is detected within an accuracy of 150 ppm and it is shown that many of the proteins in this human cancer sample are modified compared to the database. The protein M(r) map may serve as a highly reproducible standard Web-based method for comparing proteins from related human cell lines.
利用二维（2-D）液相分离/质谱图谱技术，绘制了ES2人透明细胞卵巢癌细胞蛋白质含量的分子量图谱。该方法采用从全细胞裂解物中进行蛋白质的二维液相分离，并在线连接到电喷雾电离飞行时间（ESI-TOF）质谱仪，以绘制细胞蛋白质含量的精确完整分子量（M(r)）图谱。两个分离维度包括使用液相等电聚焦作为第一阶段，以及无孔硅胶反相高效液相色谱（HPLC）作为第二阶段分离。ESI-TOF-MS检测提供了类似于二维凝胶电泳的pI与M(r)的图像。然后根据基质辅助激光解吸/电离（MALDI）-TOF-MS肽图谱和完整的M(r)对每种蛋白质进行鉴定，从而生成一个标准图谱，可用于比较其他卵巢癌细胞系。精确的完整M(r)连同pI组分和肽图谱有助于标记蛋白质，以便将来进行裂解物间的比较。还使用内标物为将来的裂解物间研究提供定量手段。在所研究的ES2细胞系中，结果表明，在pH值为4至12的17个pI组分上，以及高达85 kDa的M(r)范围内，检测到近900个M(r)条带，其中约290条带可使用基于质谱的技术进行鉴定。蛋白质M(r)的检测精度在150 ppm以内，结果表明，与数据库相比，该人类癌症样本中的许多蛋白质都发生了修饰。蛋白质M(r)图谱可作为一种基于网络的高度可重复的标准方法，用于比较相关人类细胞系中的蛋白质。
