Journal
PROTEOMICS
Volume 11, Issue 11, Pages 2308-2319Publisher
WILEY
DOI: 10.1002/pmic.201100110
Keywords
LC-MS/MS; Multidimensional LC; Protein profile; Technology
Funding
- Hong Kong Research Grants Council, Hong Kong Special Administrative Region, China [HKU7018/09P, HKU3/07C]
- Science and Technology Development Fund of Macau SAR [045/2007/A3]
- Research Committee, University of Macau [UL017]
- Hong Kong RGC
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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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