Fully automatable two-dimensional reversed-phase capillary liquid chromatography with online tandem mass spectrometry for shotgun proteomics

Proteomics
S O SiuIvan K Chu

Abstract

Herein, we describe the development of a fully automatable technology that features online coupling of high-pH RP separation with conventional low-pH RP separation in a two-dimensional capillary liquid chromatography (2-D LC) system for shotgun proteomics analyses. The complete analysis comprises 13 separation cycles, each involving transfer of the eluate from the first-dimension, high-pH RP separation onto the second RP dimension for further separation. The solvent strength increases across the 13 fractions (cycles) to elute all peptides for further resolution on the second-dimension, low-pH RP separation, each under identical gradient-elution conditions. The total run time per analysis is 52 h. In triplicate analyses of a lysate of mouse embryonic fibroblasts, we used this technology to identify 2431 non-redundant proteins, of which 50% were observed in all three replicates. A comparison of RP-RP 2-D LC and strong cation exchange-RP 2-D LC analyses reveals that the two technologies identify primarily different peptides, thereby underscoring the differences in their separation chemistries.

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Citations

Apr 25, 2012·Analytical and Bioanalytical Chemistry·Jolene K Diedrich, Ryan R Julian
Dec 6, 2012·Bioanalysis·Nm CassianoQb Cass
Jul 12, 2014·Annual Review of Analytical Chemistry·Zhaorui ZhangLjiljana Paša-Tolić
Jul 31, 2012·Journal of Chromatography. a·Fang XieYufeng Shen
Aug 24, 2013·FEBS Letters·Kazuhiro SugiharaMichiko N Fukuda
Mar 16, 2016·Scientific Data·Edward LauPeipei Ping
Jul 20, 2012·Expert Review of Proteomics·Onno KranenburgConnie R Jimenez
Aug 25, 2012·Evidence-based Complementary and Alternative Medicine : ECAM·Ming Wai HungSimon Ming Yuen Lee
Aug 17, 2021·Frontiers in Microbiology·Christina E AndronisKar-Chun Tan

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