High-efficiency on-line solid-phase extraction coupling to 15-150-μm-id column liquid chromatography for proteomic analysis

被引:96
作者
Shen, YF [1 ]
Moore, RJ [1 ]
Zhao, R [1 ]
Blonder, J [1 ]
Auberry, DL [1 ]
Masselon, C [1 ]
Pasa-Tolic, L [1 ]
Hixson, KK [1 ]
Auberry, KJ [1 ]
Smith, RD [1 ]
机构
[1] Pacific NW Natl Lab, Div Biol Sci, Richland, WA 99352 USA
关键词
D O I
10.1021/ac0300690
中图分类号
O65 [分析化学];
学科分类号
070302 ; 081704 ;
摘要
The ability to manipulate and effectively utilize small proteomic samples is important for analyses using liquid chromatography (LC) in combination with mass spectrometry (MS) and becomes more challenging for very low flow rates due to extra column volume effects on separation quality. Here we report on the use of commercial switching valves (150-mum channels) for implementing the on-line coupling of capillary LC columns operated at 10 000 psi with relatively large solid-phase extraction (SPE) columns. With the use of optimized column connections, switching modes, and SPE column dimensions, high-efficiency on-line SPE-capillary and nanoscale LC separations were obtained demonstrating peak capacities of similar to1000 for capillaries having inner diameters between 15 and 150 mum. The on-line coupled SPE columns increased the sample processing capacity by similar to400-fold for sample solution volume and similar to10-fold for sample mass. The proteomic applications of this on-line SPE-capillary LC system were evaluated for analysis of both soluble and membrane protein tryptic digests. Using an ion trap tandem MS it was typically feasible to identify 1100-1500 unique peptides in a 5-h analysis. Peptides extracted from the SPE column and then eluted from the LC column covered a hydrophilicity/hydrophobicity range that included an estimated similar to98% of all tryptic peptides. The SPE-capillary LC implementation also facilitates automation and enables use of both disposable SPE columns and electrospray emitters, providing a robust basis for automated proteomic analyses.
引用
收藏
页码:3596 / 3605
页数:10
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