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Antonino Biroccio (1, 3), Chiara Aceti (1), Andrea Urbani (2), and Giorgio Federici (1, 3)
Abstract Following electrophoretic separation of proteins the digestion is a limiting step during the process of protein identification by MS analysis. Millipore's ZipPlate* (Cat. No. ZPC1 800 10) is a 96-well plate which integrates all sample manipulations into a single unit. Therefore automated in-gel digestion using ZipPlate provides a rapid system to digest a high number of samples. For this purpose we have developed an automated method for Beckman Coulter's Biomek® 2000 Automated Laboratory Workstation. Introduction Proteomic research is rapidly scaling up in terms of the number of analyzed samples. The classic process for protein identification, including electrophoretic separation, digestion and mass spectrometry analysis, is intensive and requires many steps. We have developed an automated method for in-gel digestion using the ZipPlateT and the Multiscreen vacuum manifold (Millipore) on the Biomek 2000 workstation. By automating the in-gel digestion process, scientists can shift their efforts from sample processing to data analysis. The ZipPlate is a new 96-well plate, which incorporates multiple in-gel digestion and solid phase extraction. Each well contains a small amount of immobilized C18 resin. We present data generated with a Biomek 2000 using the ZipPlate. This automation provides a higher throughput option for protein identification and, therefore, dramatically reduces the time for protein identification compared to manual methods. Automated Procedure An automated method has been developed for the Biomek 2000 (Figure 1) for the digestion of electrophoretically separated proteins, concentration of peptide fragments in preparation for spotting on a MALDI-TOF target.
The processing of 96 samples in parallel includes all necessary plate movements, vacuum steps and liquid transfers. Proteins that have been resolved by electrophoretic separation in either one dimension or two are manually excised from the gel making sure to include only the stained gel material. The gel slices are then added to individual wells in the ZipPlate and then placed on the deck of the Biomek 2000 worksurface. The vacuum station is automatically assembled and disassembled for the elution steps using the Gripper Tool (A1, Figure 2). We have found that to ensure a proper seal for the vacuum step, the ZipPlate needs to be pushed down onto the vacuum station with the Gripper Tool.
Vacuum is applied to the plate to remove any buffer that may have been transferred to the plate along with the gel slices. Next the gel slices are destained by the addition of buffers and application of vacuum. The gel slices are then concentrated (shrunk in size) by the addition of acetonitrile. Following a short incubation the acetonitrile is removed by applying a vacuum. A trypsin enzyme solution is then added, the plate is removed from the worksurface of the Biomek 2000 for a 3-hour incubation at 37°C. (Note: this step can be automated by integration of a heating position on the worksurface of the Biomek 2000). After incubation in the trypsin solution, the plate is returned to the worksurface for the addition of acetonitrile to wet the hydrophobic C18 resin. After a short incubation at 37°C, the plate is returned to the vacuum station for the addition of the extraction/wash solution. The plate is incubated at room temperature for 30 minutes. Vacuum is then applied to extract the samples from the resin and collect for spotting onto the MALDI target. The configuration of the Biomek 2000 worksurface is reset to enable the spotting of the samples onto a MALDI target (Figure 3).
Following protein digestion and purification on ZipPlate (Millipore), peptides are automatically spotted onto a MTP (microtiter plate) Anchor Chip MALDI target and then analyzed with a MALDI-TOF-MS Bruker Reflex IV. The MALDI matrix used is α-ciano-4-hydroxycinnamic acid (CHCA) dissolved in acetonitrile/water (1:1, v/v) at a concentration of 0.5g/L. Equal volumes (1.5µL) of matrix and analyte solutions are mixed and 2 µL of the mixture is deposited on the MALDI target and dried under ambient conditions. Samples are recrystallized, applying 0.5µL of a recrystallization solution (EtOH:Acetone:0.1% Trifluor acetic acid; ratio 6:3:1). Samples are dried at room temperature before MALDI-TOF-MS. The use of the Biomek 2000 tools to spot the samples onto the target (Bruker Daltonics) is shown in Figure 1. For best results for spotting, we have found that maintaining a dust-free environment is essential. For this reason we strongly recommend that the Biomek 2000 system is placed in a standard sterile hood.
Results and Discussion Once the samples are spotted onto the MALDI target the peptides were analyzed using a MALDI-TOF Reflex IV system from Bruker Daltonics. The excellent results are showing high sequence coverage (Fig. 4). We have automated the in-gel digestion system for rapid digestion of proteins derived from an electrophoretic run. The innovative system eliminates the need for manual intervention and allows minimization of the protein identification time. This protocol was developed for protein 2D-PAGE spots stained with blue colloidal coomassie. Similar results with a further reduced process time can be obtained from silver stained gels. The automated in-gel protein digestion and MS sample preparation developed for the Biomek 2000 platform allows a quick startup in our laboratory and offers several key advantages for end-users. The simple processing of several proteins at the same time, removes a former bottleneck in protein identification. Acknowledgements We are particularly thankful to Dott. Carlo Raviolo (Millipore Inc.) for technical support. * All trademarks are the property of their respective owners. For Research Use Only; not for use in diagnostic procedures.
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