Pre-Fractionation of Highly Complex Proteomic Samples Using the UltiMate 3000 Proteomics MDLC

Multidimensional LC coupled to mass spectrometry is a valuable technique in both top-down and bottom-up proteomic workflows . Offline Multidimensional LC offers several advantages over online approaches:

• High flexibility in column dimensions and mobile phase selection,
• Simple method development and optimization of each separation dimension,
• Ability to re-analyze sample fractions.

Schematic setup for automated offline 2-D LC of peptides.

The typical workflow for automated offline MDLC involves the following steps:

1) Sample injection
2) First-dimension LC separation with fraction collection
3) Re-injection of the collected fractions
4) Second-dimension LC separation
5) Detection of peptides by MS/MS or MSn.

Visualization of 2-D LC data in Chromeleon: Top panel: First-dimension separation; Middle panel 2-D retention map;  Lower panel: Second-dimension separation.

Related Documents

• Fully Automated Off-line Multidimensional LC Methods in Proteomics (HPLC 2006, 0.5MB)
• Combined Injection and Fractionation Capabilities of an Autosampler for Automated Off-Line Multidimensional LC Methods in Proteomics (Pittcon 2006, 0.7MB)
• Proteome Analysis Involving Off-Line 2-D LC of Intact Proteins, Proteolytic Digestion, and Capillary RP-LC-MS/MS Analysis Using Monolithic PS-DVB Columns (AN 530, 0.7MB)