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. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. 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. Protein standards were digested in-solution on-target/in-well using the BD MALDI Concentrator, dried under vacuum and co-crystallized with matrix under differing conditions. 1D and 2D-HPLC fractionation of protein mixtures was conducted with a Beckman PF2D system. Fractions were collected directly into the wells of the BD device, and optimized conditions were used to concentrate, digest in-solution on-target/in-well, and co-crystallize the samples with matrix. MALDI mass spectra were obtained with a Bruker Reflex IV MALDI-TOF MS. Results were compared, with fractionated protein mixtures and peptide standards that had been digested, concentrated and co-crystallized with matrix by conventional methods. Results were further compared with LC fraction collection into 96 well plates and with 1D SDS-PAGE followed by in-gel digestion of proteins. One-pot on-target/in-well digestion, concentration and sample/matrix co-crystallization under optimized solvent conditions readily yielded MS analyses with minimal sample loss from 1 pmol protein standards and as little as 10 fmol of peptide standards from up to 200 ¿l starting solution. This amounted to good recovery of MS signal from pMol protein and sub pMol peptide concentrations. This methodology was expanded to analyze protein mixtures separated by 1D and 2D RP-protein-LC. Results using the BD Concentrator compared well with in-gel digestion of protein standards separated via SDS-PAGE and with standards separated by 1D and 2D RP-protein-LC. While the resolution of the current RP-LC separation is less than that obtained with 1D SDS-PAGE, the ease and degree of recovery is enhanced as is the ability to automate the system. 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 enabled rapid, high-throughput protein fractionation, digestion, peptide matrix co-crystallization, and MALDI-TOF MS analyses with minimal sample handling. Three manuscripts resulting from this project were published this year.