2D-LC MALDI WITH ON-TARGET DIGESTION FOR HIGH-THROUGHPUT PROTEOMICS

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Large-scale proteomic analyses necessitate high-throughput sample preparation techniques. However, highly complex mixtures require multi-dimensional fractionation prior to MS analysis to maximize the yield of useful MS data. This geometrically expands sample numbers, dramatically intensifying processing load, commonly involves dilution (e.g., HPLC), often demanding sample concentration, and typically requires multiple steps of sample handling, including transfer to different reaction vessels. These steps are time consuming, lead to sample losses and potential contamination. We have explored the use of a novel, simple, inexpensive (non-robotic) 96-well array technology, the BD MALDI Concentrator, to conduct one-pot on-target sample preparation for MALDI-MS analysis. We have applied this technology with deposition from 1D and 2D protein LC direct-to-target for peptide mapping by MALDI-TOF MS. 1D and 2D-HPLC fractionation of protein mixtures is conducted with a Beckman PF2D system. Fractions can be collected directly into the wells of the BD device, and optimized conditions are used to concentrate, digest in-solution on-target/in-well, and co-crystallize the samples with matrix. MALDI mass spectra are obtained with a Bruker Reflex IV MALDI-TOF MS. Results are compared to other sample handling methods. One-pot on-target/in-well digestion, concentration and sample/matrix co-crystallization under optimized solvent conditions readily yields good quality mass spectra from as little as 10 fmol of peptide standards from up to 200 ¿l starting solution. The coupling of 1D and 2D-protein-LC to MALDI-TOF MS through the collection of LC fractions directly into the 96-well array concentrator enables rapid, high-throughput protein fractionation, digestion, peptide matrix co-crystallization, and MALDI-TOF MS analyses with minimal sample handling. The method is now being applied to proteomic projects of the Resource and the CPC and the results are being evaluated to generate further refinements of the procedures.