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Fully automatable two‐dimensional reversed‐phase capillary liquid chromatography with online tandem mass spectrometry for shotgun proteomics
Author(s) -
Siu S. O.,
Lam Maggie P. Y.,
Lau Edward,
Kong Ricky P. W.,
Lee Simon M. Y.,
Chu Ivan K.
Publication year - 2011
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/pmic.201100110
Subject(s) - chromatography , shotgun proteomics , chemistry , elution , tandem mass spectrometry , proteomics , mass spectrometry , resolution (logic) , shotgun , liquid chromatography–mass spectrometry , lysis , analytical chemistry (journal) , biochemistry , artificial intelligence , computer science , gene
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.