Preparation of 20-μm-i.d. silica-based monolithic columns and their performance for proteomics analyses
Preparation of 20-μm-i.d. silica-based monolithic columns and their performance for proteomics analyses
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DOI：
10.1021/ac050454k
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发表时间：
2005-08-01
影响因子：
7.4
通讯作者：
Smith, RD
中科院分区：
文献类型：
--
作者：
Luo, QZ;Shen, YF;Smith, RD
我们描述了高效 70 cm x 20 mu m i.d. 的制备和性能。硅胶基整体式毛细管液相色谱柱。流动相压力为 5000 psi 的整体柱可提供类似于 40 nL/min 的流速和类似于 0.24 cm/s 的线速度。该色谱柱与微固相萃取在线耦合和纳电喷雾电离质谱联用，提供类似于 420 的分离峰容量。使用 Shewanella oneidensis 胰蛋白酶消化物评估性能，并使用传统离子阱和 MS/MS 进行肽鉴定，获得类似于 15-amol 的肽检测限。灵敏度和分离效率使得能够在单次 10 小时分析中从 2.5 μg 胰蛋白酶消化样品中鉴定出 2367 种不同的肽，涵盖 855 种不同的 S. oneidensis 蛋白质。当有效分离时间从 200 分钟延长至 600 分钟时，鉴定出的肽和蛋白质的数量大约增加了一倍。随着注射量从 0.5 ng 增加到 100 ng，鉴定出的肽数量从 32 增加到 390。还评估了蛋白质组分析的运行间和柱间重现性。
We describe the preparation and performance of high-efficiency 70 cm x 20 mu m i.d. silica-based monolithic capillary LC columns. The monolithic columns at a mobile-phase pressure of 5000 psi provide flow rates of similar to 40 nL/min at a linear velocity of similar to 0.24 cm/s. The columns provide a separation peak capacity of similar to 420 in conjunction with both on-line coupling with microsolid-phase extraction and nanoelectrospray ionization-mass spectrometry. Performance was evaluated using a Shewanella oneidensis tryptic digest, and similar to 15-amol detection limits for peptides were obtained using a conventional ion trap and MS/MS for peptide identification. The sensitivity and separation efficiency enabled the identification of 2367 different peptides covering 855 distinct S. oneidensis proteins from a 2.5-mu g tryptic digest sample in a single 10-h analysis. The number of identified peptides and proteins approximately doubled when the effective separation time was extended from 200 to 600 min. The number of identified peptides increased from 32 to 390 as the injection amount was increased from 0.5 to 100 ng. Both the run-to-run and column-to-column reproducibility for proteomic analyses were also evaluated.