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2D Separations
The Eksigent NanoLC™-2D system enables the user to choose step or linear gradient elution mode as part of an on-line 2D LC/MS/MS analysis with both modes being equally successful in identifying peptides.
On-line two-dimensional (2D) chromatography is often used to increase peak capacity for the analysis of complex peptide samples by LC/MS/MS. Increased separation of the peptides typically results in a higher number of identified peptides in a "shotgun" approach proteomics experiment. A Strong Cation Exchange (SCX) column is commonly used for the first dimension separation in combination with a Reversed Phase (RP) column in the second dimension. Peptides can be eluted by stepwise changing the ionic strength of the eluent in the first dimension, collecting peptides on a trap and then subjecting them to reversed phase chromatography in the second dimension. Alternatively, peptides can be eluted from the ion exchange column with a continuous or discontinuous linear gradient.
The ability of the Eksigent NanoLC-2D to operate in a variety of modes without requiring changes to the hardware configuration has allowed us to investigate the effect of elution mode on peak shape and peptide identification while keeping other parameters constant. Studies in the literature have reported broad peaks or fewer identified peptides with step gradient elution in the SCX dimension. However, these studies often overlooked other contributing factors such as elution time which also plays a critical role in on-line SCX-RP analyses. In this study, step and linear gradient SCX elution modes are compared for an on-line 2D nano LC/MS/MS analysis using a five-protein digest. Contrary to past reports, step elution mode performed at least as well as and possibly better than linear elution mode for the identification of peptides in this application. Step and linear gradient elution modes were equally successful in identifying an adequate number of peptides to represent the proteins in the original sample.
The Eksigent NanoLC-2D system allows flexibility for the user to choose from a variety of possible ion exchange elution modes as part of an on-line 2D LC/MS/MS analysis.