On-line 1D and 2D porous layer open tubular/LC-ESI-MS using 10-μm-i.d. poly(styrene-divinylbenzene) columns for ultrasensitive proteomic analysis
On-line 1D and 2D porous layer open tubular/LC-ESI-MS using 10-μm-i.d. poly(styrene-divinylbenzene) columns for ultrasensitive proteomic analysis
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DOI：
10.1021/ac070583w
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发表时间：
2007-08-15
影响因子：
7.4
通讯作者：
Karger, Barry L.
中科院分区：
文献类型：
--
作者：
Luo, Quanzhou;Yue, Guihua;Karger, Barry L.
继我们最近的工作之后，使用 3.2 m x 10 mu m i.d. 的在线一维 (1D) 和二维 (2D) 多孔层开放管/液相色谱-电喷雾电离-质谱 (PLOT/LC-ESI-MS) 平台。聚（苯乙烯-二乙烯基苯）(PS-DVB) PLOT 色谱柱已开发用于提供稳健、高性能和超灵敏的蛋白质组分析。通过使用 PicoClear T 形接头，内径为 50,μm 的管之间的死体积连接可以实现。 PS-DVB整体式微型SPE柱和PLOT柱被最小化。 microSPE/PLOT 柱组件提供的分离性能类似于以 20 nL/min 的流动相流速直接注射到 PLOT 柱上所获得的分离性能。使用宫颈癌 (SiH α) 细胞系凝胶组分 (15-40 kDa) 的凝胶内胰蛋白酶消化样品评估该平台的痕量分析潜力。作为系统灵敏度的一个例子，类似于 2 μL 溶液中的 2.5 ng 蛋白质，相当于 20 个 SiHa 细胞的量，使用线性离子阱 MS 进行在线微型 SPE-PLOT/LC-ESI-MS/MS 分析。当使用假阳性率低于 1% 的严格标准时，通过单次分析共鉴定出与 163 种独特蛋白质相关的 237 种肽。当注射量增加到大约 45 ng 蛋白质（相当于 350 个 SiHa 细胞）时，鉴定出的肽和蛋白质的数量分别增加到 638 和 343。相比之下，使用典型的 15 cm x 75 lint i.d.，仅从来自相同凝胶组分的 750 ng 蛋白质（相当于 6000 个 SiHa 细胞）中鉴定出 338 种肽和 231 种独特蛋白质（假阳性率再次低于 1%）。填充毛细管柱。 PLOT 色谱柱具有更高的灵敏度、更高的回收率和更高的分辨能力，导致鉴定数量从仅进样样品量的 5% 左右增加。通过将高效反相 PLOT 柱与强正则交换色谱 (SCX) 相结合，二维色谱进一步扩展了微型 SPE/PLOT 组件的分辨率。例如，在在线 2D SCX-PLOT/LC-MS 中使用五个离子交换组分，从来自同一凝胶组分的 75 ng 蛋白质（相当于 600 个细胞）中鉴定出与 536 种独特蛋白质相关的 1071 个肽。以自动化格式实施的 2D 系统使蛋白质组分析操作简单而稳健。这些有希望的结果证明了 PLOT 柱用于超痕量分析的潜力。
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 m x 10 mu m 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,mu m i.d. PS-DVB monolithic micro-SPE column and the PLOT column was minimized. The microSPE/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 (SiH alpha) cell line. As an example of the sensitivity of the system, similar to 2.5 ng of protein in 2,mu L 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 similar to 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 cm x 75 lint 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 similar to 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 canon-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.