Luo Quanzhou, Yue Guihua, Valaskovic Gary A, Gu Ye, Wu Shiaw-Lin, Karger Barry L
Barnett Institute and Department of Chemistry and Chemical Biology, Northeastern University, Boston, Massachusetts 02115, USA.
Anal Chem. 2007 Aug 15;79(16):6174-81. doi: 10.1021/ac070583w. Epub 2007 Jul 11.
Following on our recent work, on-line one-dimensional (1D) and two-dimensional (2D) porous layer open tubular/liquid chromatography-electrospray ionization-mass spectrometry (PLOT/LC-ESI-MS) platforms using 3.2 mx10 microm i.d. poly(styrene-divinylbenzene) (PS-DVB) PLOT columns have been developed to provide robust, high-performance, and ultrasensitive proteomic analysis. With the use of a PicoClear tee, the dead volume connection between a 50 microm i.d. PS-DVB monolithic micro-SPE column and the PLOT column was minimized. The micro-SPE/PLOT column assembly provided a separation performance similar to that obtained with direct injection onto the PLOT column at a mobile phase flow rate of 20 nL/min. The trace analysis potential of the platform was evaluated using an in-gel tryptic digest sample of a gel fraction (15-40 kDa) of a cervical cancer (SiHa) cell line. As an example of the sensitivity of the system, approximately 2.5 ng of protein in 2 microL of solution, an amount corresponding to 20 SiHa cells, was subjected to on-line micro-SPE-PLOT/LC-ESI-MS/MS analysis using a linear ion trap MS. A total of 237 peptides associated with 163 unique proteins were identified from a single analysis when using stringent criteria associated with a false positive rate of less than 1%. The number of identified peptides and proteins increased to 638 and 343, respectively, as the injection amount was raised to approximately 45 ng of protein, an amount corresponding to 350 SiHa cells. In comparison, only 338 peptides and 231 unique proteins were identified (false positive rate again less than 1%) from 750 ng of protein from the identical gel fraction, an amount corresponding to 6000 SiHa cells, using a typical 15 cmx75 microm i.d. packed capillary column. The greater sensitivity, higher recovery, and higher resolving power of the PLOT column resulted in the increased number of identifications from only approximately 5% of the injected sample amount. The resolving power of the micro-SPE/PLOT assembly was further extended by 2D chromatography via combination of the high-efficiency reversed-phase PLOT column with strong cation-exchange chromatography (SCX). As an example, 1071 peptides associated with 536 unique proteins were identified from 75 ng of protein from the same gel fraction, an amount corresponding to 600 cells, using five ion-exchange fractions in on-line 2D SCX-PLOT/LC-MS. The 2D system, implemented in an automated format, led to simple and robust operation for proteomic analysis. These promising results demonstrate the potential of the PLOT column for ultratrace analysis.
基于我们最近的工作，已开发出使用内径为3.2毫米×10微米的聚（苯乙烯 - 二乙烯基苯）（PS - DVB）多孔层开管柱的在线一维（1D）和二维（2D）多孔层开管/液相色谱 - 电喷雾电离 - 质谱（PLOT/LC - ESI - MS）平台，以提供强大、高性能和超灵敏的蛋白质组学分析。通过使用PicoClear三通，将内径为50微米的PS - DVB整体式微固相萃取柱与PLOT柱之间的死体积连接降至最低。微固相萃取/PLOT柱组件在20纳升/分钟的流动相流速下，提供了与直接注入PLOT柱相似的分离性能。使用宫颈癌（SiHa）细胞系凝胶组分（15 - 40 kDa）的胶内胰蛋白酶消化样品评估了该平台的痕量分析潜力。作为系统灵敏度的一个例子，对2微升溶液中约2.5纳克蛋白质（相当于20个SiHa细胞的量）使用线性离子阱质谱进行在线微固相萃取 - PLOT/LC - ESI - MS/MS分析。当使用与假阳性率小于1%相关的严格标准时，单次分析共鉴定出与163种独特蛋白质相关的237种肽。随着进样量增加到约45纳克蛋白质（相当于350个SiHa细胞的量），鉴定出的肽和蛋白质数量分别增加到638和343。相比之下，使用典型的15厘米×75微米内径的填充毛细管柱，从相同凝胶组分的750纳克蛋白质（相当于6000个SiHa细胞的量）中仅鉴定出338种肽和231种独特蛋白质（假阳性率同样小于1%）。PLOT柱更高的灵敏度、更高的回收率和更高的分离能力使得仅从约5%的进样量中就能鉴定出更多的蛋白质。通过将高效反相PLOT柱与强阳离子交换色谱（SCX）相结合的二维色谱法，进一步扩展了微固相萃取/PLOT组件的分离能力。例如，使用在线二维SCX - PLOT/LC - MS中的五个离子交换馏分，从相同凝胶组分的75纳克蛋白质（相当于600个细胞的量）中鉴定出与536种独特蛋白质相关的1071种肽。以自动化形式实施的二维系统为蛋白质组学分析带来了简单且强大的操作。这些有前景的结果证明了PLOT柱在超痕量分析中的潜力。
