RESUMO
The combination of gel-based two-dimensional protein separations with protein identification by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) is the workhorse for the large-scale analyses of proteomes. Such high-throughput proteomic approaches require automation of all post-separation steps and the in-gel digest of proteins especially is often the bottleneck in the protein identification workflow. With the objective of reaching the same high performance of manual low-throughput in-gel digest procedures, we have developed a novel stack-type digestion device and implemented it into a commercially available robotic liquid handling system. This modified system is capable of performing in-gel digest, extraction of proteolytic peptides, and subsequent sample preparation for MALDI-MS without any manual intervention, but with a performance at least identical to manual procedures as indicated on the basis of the sequence coverage obtained by peptide mass fingerprinting. For further refinement of the automated protein identification workflow, we have also developed a motor-operated matrix application device to reproducibly obtain homogenous matrix preparation of high quality. This matrix preparation was found to be suitable for the automated acquisition of both peptide mass fingerprint and fragment ion spectra from the same sample spot, a prerequisite for high confidence protein identifications on the basis of peptide mass and sequence information. Due to the implementation of the stack-type digestion device and the motor-operated matrix application device, the entire platform works in a reliable, cost-effective, and sensitive manner, yielding high confidence protein identifications even for samples in the concentration range of as low as 100 fmol protein per gel plug.
