This Application Note describes a method for increasing the resolution of complex proteome samples by using two-dimensional offline LC/MS. The method applies a continuous salt gradient in the first separation dimension, which can significantly increase the number of identified proteins from complex samples due to higher chromatographic resolution compared to stepwise elution. In addition, the method offers high flexibility for the investigator since micro fractions of the first dimension can be stored, re-analyzed or chemically modified prior to further analysis. To achieve optimal resolution, the applied 2D LC method strongly requires optimization of the two separation dimensions. Especially elution gradients, analysis time, stationary phase material, and column dimension need to be designed and assembled adequately to attain a considerable increase in overall peak capacity. This Application Note shows data which influences the variables and components. It can serve as a guideline for the investigator to adapt his separation method for a specific proteome sample in order to achieve maximum peptide sequence information by MS/MS analysis. This will result in a maximum of identified proteins after a database search.
This document outlines a method for analyzing highly complex proteomic samples using off-line two-dimensional high-performance liquid chromatography (2D-HPLC). The technique combines strong cation exchange (SCX) separation with reversed phase (RP) chromatography to significantly increase the number of identified proteins compared to stepwise elution methods. By applying a continuous salt gradient in the first dimension, the method achieves higher chromatographic resolution and offers flexibility for reanalysis or chemical modification of micro fractions.
A continuous salt gradient significantly increases the number of identified proteins from complex samples due to higher chromatographic resolution compared to stepwise elution.
The Agilent BioSCX Series II column has a dimension of 0.30 x 35 mm and uses 3.5 um particles.
The maximum accumulation time for the MS scan is set to 150 ms.