RP-LC x RP-LC analysis of a tryptic digest using a combination of totally porous and partially porous stationary phases
RP-LC x RP-LC analysis of a tryptic digest using a combination of totally porous and partially porous stationary phases
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DOI：
10.1002/jssc.200900816
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发表时间：
2010-05-01
影响因子：
3.1
通讯作者：
Dugo, Paola
中科院分区：
文献类型：
--
作者：
Mondello, Luigi;Donato, Paola;Dugo, Paola
不同的色谱分离模式已经结合，在离线和在线的方法，试图实现提高分离能力，以分析复杂的蛋白质组样品。其中，RP-LC x RP-LC的使用带来了两个维度的高峰值容量，完全自动化的能力，以及与ms直接连通性的可能性的附加优势。在这一贡献中，我们通过在碱性pH，中等压力下串行耦合生物宽孔C18柱，在第一个维度上实现了增强的分辨率。选择性使用一个短（3厘米）C18柱填充部分多孔颗粒，用于快速二维分析，在酸性ph下操作。综合系统配置在电子激活的两个位置，10端口阀周围，用于循环内自动馏分收集/再进。理论峰值容量为3796计算的设置采用一个基本柱在第一个维度，而峰值容量为10686达到了四倍的固定相的长度，即使在较长的分析时间的代价。系统重现性好，保留时间和色谱峰面积的RSD分别小于1.2和4%。
Different chromatographic separation modes have been combined, in off-line and on-line approaches, attempting to achieve enhanced separation power for the analysis of complex proteome samples. Among these, the use of RP-LC x RP-LC brings in the added advantages of high peak capacity in both dimensions, capability of full automation, and the likelihood of direct hyphenation to MS. In this contribution, we achieved enhanced resolution in the first dimension, by serially coupling Bio wide pore C18 columns at basic pH, at moderate pressure. Selectivity was tuned using one short (3 cm) C18 column packed with partially porous particles for fast second-dimension analysis, operated at acidic pH. The comprehensive system was configured around electronically activated two-position, ten-port valve for within-loop automated fraction collection/re-injection. A theoretical peak capacity of 3796 was calculated for the set-up employing one basic column in the first dimension, while a peak capacity of 10 686 was attained with four times the length of stationary phase, even at the expense of longer analysis time. The system showed very good reproducibility of retention times and chromatographic peak areas (RSD less than 1.2 and 4%, respectively).