Abstract
2D HPLC separations by coupling strong cation exchange (SCX) and RP fractionation have been widely used in large-scale proteomic studies. Traditionally this method is performed by salt gradient SCX separation followed by RP and MS/MS analysis. The salt gradient SCX method has been known to have low peptide and protein resolution. In this study, we implemented a pH gradient SCX-RP HPLC platform to separate proteome digests from adult zebrafish hearts, followed by ESI quadrupole-TOF MS/MS analysis. This pH gradient SCX method has improved peptide separation, as demonstrated by a greater number of peptides and proteins identified from individual SCX fractions. This pH gradient method also has better MS compatibility owing to lower salt usage. This setup allows fast microflow fractionation in SCX dimension and nanoflow RP separation in the second dimension, and can be easily implemented on conventional capillary LC ESI MS/MS systems. Using this setup, we identified 1375 proteins from adult zebrafish hearts, establishing the first reported experimental data set for the heart proteome of zebrafish. This work laid the foundation for further studies of environmental cardiac toxicology using zebrafish as a model organism.
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