Improved 2D nano-LC/MS for proteomics applications: a comparative analysis using yeast proteome

The most commonly used method for protein identification with two-dimensional (2D) online liquid chromatography-mass spectrometry (LC/MS) involves the elution of digest peptides from a strong cation exchange column by an injected salt step gradient of increasing salt concentration followed by reversed phase separation. However, in this approach ion exchange chromatography does not perform to its fullest extent, primarily because the injected volume of salt solution is not optimized to the SCX column. To improve the performance of strong cation exchange chromatography, we developed a new method for 2D online nano-LC/MS that replaces the injected salt step gradient with an optimized semicontinuous pumped salt gradient. The viability of this method is demonstrated in the results of a comparative analysis of a complex tryptic digest of the yeast proteome using the injected salt solution method and the semicontinuous pump salt method. The semicontinuous pump salt method compares favorably with the commonly used injection method and also with an offline 2D-LC method.