Proteomics Workflow With the UltiMate 3000

Purification, identification, and characterization of proteins from cell lysates are key challenges in proteomic research. Workflows used to achieve this goal are :

1. Prepare a purification sample from cell lysates.
2. Perform protein purification with multidimensional chromatography using a mixture of ion-exchange and reverse-phase columns.
3. Digest purified fractions with an enzyme such as trypsin.
4. Perform peptide purification with multidimensional chromatography using a mixture of ion-exchange and reverse-phase columns.
5. Identify and characterize peptides (and by association the original protein) in final fractions using a nano-LC combined with an MS detector.

The total number of steps and separation dimensions as well as the types of columns involved is dependent on the number of proteins in the sample. For example, if there is a very small number of proteins in the original sample it is possible to start directly at the digestion stage.

Intelligent LC solutions provided by the powerful UltiMate ^TM 3000 system combined with Dionex column technology and the Chromeleon^® Chromatography Management Software allow the complete automation of most of these steps and provides the following benefits:

• Unique x2 dual-gradient systems enable ion-exchange AND reverse-phase separation on one system.
• The advanced UltiMate 3000 well plate sampler can both inject and fractionate, providing fully automated off-line 2-D LC and protein prefractionation capabilities.
• UltiMate 3000 also provides nanoflow capabilities for the final analytical stage.
• On-line fraction collection on MALDI targets with the Dionex Probot ^TM .
• Complete range of column types for your separation needs.
• Simple yet powerful purification control with the Chromeleon Chromatography Management Software.
• DCMS^Link provides fully integrated, single-point control of UltiMate 3000 through Xcalibur ^TM  (Thermo Fisher), Analyst^® (AB/Sciex) and HyStar™ (Bruker Daltonics) for easy ESI coupling.
• Most steps in the process can be fully automated with UltiMate 3000, leading to higher sample throughput, more reliable results, and improved laboratory productivity.

Three predefined configurations of the UltiMate 3000 are available for this kind of workflow. These configurations come with all modules, tubing, and software features that are required:

• UltiMate 3000 LC Salt Plug Configuration—for the analysis of moderately complex samples.
• UltiMate 3000 Comprehensive On-Line 2-D LC Configuration—for the analysis of complex samples.
• Ultimate 3000 Fully Automated Off-Line 2-D LC Configuration—for maximum versatility in the analysis of complex samples.

All three configurations are available as biocompatible systems.