The UltiMate^® 3000 Proteomics MDLC system, which is based on the RSLCnano system, is the essential tool for multidimensional LC separations of highly complex biological samples. The dual-gradient RSLCnano system provides two independent, precise gradients. The system is highly flexible, supporting a wide range of MDLC applications.

• Wide flow rate range: 20 nL/min–2.5 mL/min
• Dual-gradient pump with two independent gradient pumps with superior gradient accuracy and precision
• Easily convertable between nano, capillary, and micro flow rates
• Continuous direct flow for interruption free analysis
• Low volume nano/cap/micro flow cells for UV-vis detector
• Micro fraction collection option for fully automated off-line MDLC
• Supports key solutions such as automated off-line 2D-LC and 2D-LC using salt plugs

The UltiMate 3000 Proteomics MDLC system supports orthogonal separation techniques such as ion-exchange and reversed-phase chromatography in on-line and off-line 2D-LC mode. It is the ideal LC system for bioanalytical laboratories that want to profit from automated sample preparation and higher sensitivity.

UltiMate 3000 Proteomics MDLC Configurations, Applications and Column Technology.

On-line MDLC strategy: well-matched for further separation of digested 1-D gel bands or 2-D gel spots. In addition, this approach provides excellent results for the analysis of highly complex proteomic samples, such as purified biological fluids or PTM complexes.

Fully Automated Off-line MDLC strategy: specifically designed for highly complex proteomic samples such as cell lysates and tissues. Using the advanced technology of the WPS-3000 well plate autosampler, sample pre-fractionation can be performed, and then followed by re-injection of each fraction without any manual handling of the sample. This automated approach to sample preparation allows for the identification of low-abundance proteins or biomarkers.

The UltiMate 3000 Proteomics MDLC system provides:

• Unique x2 dual-gradient systems that enables multidimensional separation on one system
• The advanced UltiMate 3000 well plate sampler for both injection and fractionation, providing fully automated off-line 2D-LC and protein pre-fractionation
• Nanoflow capabilities for the final analytical stage
• On-line fraction collection on MALDI targets with the Probot™ Microfraction Collector/Spotter
• Complete range of column types for your separation needs
• Fractionation control and visualization of 2-D retention map using Chromeleon^® Chromatography Management Software
• DCMS^Link™ for single-point control of UltiMate 3000 through Xcalibur™ (Thermo Fisher), Analyst^®  (AB/Sciex) and HyStar™ (Bruker Daltonics) for easy ESI coupling
• Easy automation of separation processes, leading to higher sample throughput, more reliable results, and improved laboratory productivity

Pre-Fractionation

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.

Workflow

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

• Preparing a purification sample from cell lysates
• Performing protein purification with multidimensional chromatography using a mixture of ion-exchange and reversed-phase columns
• Digesting purified fractions with an enzyme such as trypsin
• Performing peptide purification with multidimensional chromatography using a mixture of ion-exchange and reversed-phase columns
• Identifying and characterizing 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^® 3000 system combined with Dionex column technology and the Chromeleon^® Chromatography Data System software allow the complete automation of most of these steps and provides the following benefits:

• Unique x2 dual-gradient systems enable ion-exchange and reversed-phase separation on one system.
• The advanced UltiMate 3000 well plate sampler can both inject and fractionate, providing fully automated off-line 2D-LC and protein prefractionation capabilities.
• UltiMate 3000 also provides nano flow capabilities for the final analytical stage.
• On-line fraction collection on MALDI targets with the Dionex Probot^™.
• Complete range of column types for your separation needs.
• Simple yet powerful purification control with the Chromeleon Chromatography Data System software.
• DCMS^Link™ provides fully integrated, single-point control of UltiMate 3000 through Xcalibur^™ (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.

The following LC modules and accessories represent a typical Ultimate 3000 Proteomics MDLC system configuration. It is the most flexible system for preconcentration and proteomics MDLC solutions such as automated off-line 2-D LC and 2-D LC salt plug.