Capillary Array Reversed-Phase Liquid Chromatography-Based Multidimensional Separation System Coupled with MALDI-TOF-TOF-MS Detection for High-Throughput Proteome Analysis
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- 作者：Xue Gu ; Chunhui Deng ; Guoquan Yan ; Xiangmin Zhang
- 刊名：Journal of Proteome Research
- 出版年：2006
- 出版时间：November 2006
- 年：2006
- 卷：5
- 期：11
- 页码：3186 - 3196
- 全文大小：821K
- 年卷期：v.5,no.11(November 2006)
- ISSN：1535-3907
文摘
A high-throughput on-line capillary array-basedtwo-dimensional liquid chromatography (2D-LC) systemcoupled with MALDI-TOF-TOF-MS proteomics analyzerfor comprehensive proteomic analyses has been developed, in which one capillary strong-cation exchange (SCX)chromatographic column was used as the first separationdimension and 18 parallel capillary reversed-phase liquidchromatographic (RPLC) columns were integrated as thesecond separation dimension. Peptides bound to the SCXphase were "stepped" off using multiple salt pulsesfollowed by sequentially loading of each subset of peptides onto the corresponding precolumns. After saltfractionation, by directing identically split solvent-gradientflows into 18 channels, peptide fractions were concurrently back-flushed from the precolumns and separatedsimultaneously with 18 capillary RP columns. LC effluentswere directly deposited onto the MALDI target platesthrough an array of capillary tips at a 15-s interval, andthen 
-cyano-4-hydroxycinnamic acid (CHCA) matrix solution was added to each sample spot for subsequentMALDI experiments. This new system allows an 18-foldincrease in throughput compared with serial-based 2D-LC system. The high efficiency of the overall system wasdemonstrated by the analysis of a tryptic digest ofproteins extracted from normal human liver tissue. A totalof 462 proteins was identified, which proved the system'spromising potential for high-throughput analysis andapplication in proteomics.
-cyano-4-hydroxycinnamic acid (CHCA) matrix solution was added to each sample spot for subsequentMALDI experiments. This new system allows an 18-foldincrease in throughput compared with serial-based 2D-LC system. The high efficiency of the overall system wasdemonstrated by the analysis of a tryptic digest ofproteins extracted from normal human liver tissue. A totalof 462 proteins was identified, which proved the system'spromising potential for high-throughput analysis andapplication in proteomics.