Kachman Maureen T, Wang Haixing, Schwartz Donald R, Cho Kathleen R, Lubman David M
Department of Chemistry, School of Medicine, The University of Michigan, Ann Arbor 48109-1055, USA.
Anal Chem. 2002 Apr 15;74(8):1779-91. doi: 10.1021/ac011159c.
A two-dimensional liquid phase separation of proteins from whole cell lysates coupled on-line to an electrospray-ionization time-of-flight (ESI-TOF) mass spectrometer (MS) is used to map the protein content of ovarian surface epithelial cells (OSE) and an ovarian carcinoma-derived cell line (ES2). The two dimensions involve the use of liquid isoelectric focusing as the first phase and nonporous silica reversed-phase HPLC as the second phase of separation. Accurate molecular weight (MW) values are then obtained upon the basis of ESI-TOFMS so that an image of isolectric point (pI) versus MW analogous to 2-D gel electrophoresis is produced. The accurate MW together with the pI fraction and corresponding hydrophobicity (%B) are used to tag each protein so that protein expression can be compared in interlysate studies. Each protein is also identified on the basis of matrix-assisted laser desorption-ionization (MALDI) TOFMS peptide mapping and intact MW so that a standard map is produced against which other cell lines can be compared. Quantitative changes in protein expression are measured in these interlysate comparisons using internal standards in the on-line ESI-TOFMS process. In the ovarian epithelial cell lines under study, it is shown that in the three pI fractions chosen for detailed analysis, over 50 unique proteins can be detected per fraction, of which 40% can be identified from web-based databases. It is also shown that when using an accurate MW to compare proteins in the OSE versus ovarian cancer sample, there are proteins highly expressed in cancer cells but not in normal cells. In addition, many of the proteins in the cancer sample appear to be down-regulated, as compared to the normal cells. This two-dimensional (2-D) liquid/mass mapping method may provide a means of studying proteins in interlysate comparisons not readily available by other methods.
将全细胞裂解物中的蛋白质进行二维液相分离，并在线连接到电喷雾电离飞行时间（ESI-TOF）质谱仪（MS）上，用于绘制卵巢表面上皮细胞（OSE）和一种卵巢癌衍生细胞系（ES2）的蛋白质图谱。这两个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作为第二阶段的分离。然后基于ESI-TOFMS获得准确的分子量（MW）值，从而生成类似于二维凝胶电泳的等电点（pI）对MW的图像。准确的MW连同pI分数和相应的疏水性（%B）用于标记每种蛋白质，以便在裂解物间研究中比较蛋白质表达。每种蛋白质还基于基质辅助激光解吸电离（MALDI）TOFMS肽图谱和完整的MW进行鉴定，从而生成一个标准图谱，可用于与其他细胞系进行比较。在在线ESI-TOFMS过程中使用内标物，在这些裂解物间比较中测量蛋白质表达的定量变化。在所研究的卵巢上皮细胞系中，结果表明，在选择用于详细分析的三个pI分数中，每个分数可检测到超过50种独特的蛋白质，其中40%可从基于网络的数据库中鉴定出来。还表明，当使用准确的MW比较OSE与卵巢癌样本中的蛋白质时，存在在癌细胞中高表达但在正常细胞中不表达的蛋白质。此外，与正常细胞相比，癌样本中的许多蛋白质似乎下调了。这种二维（2-D）液相/质谱图谱方法可能提供一种在裂解物间比较中研究蛋白质的手段，而其他方法不易获得这种手段。
