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.
Specific Aim 1 for this annual report focuses on developments associated with automated analysis, proteome sample processing, and new developments with field asymmetric waveform ion mobility spectrometry analyzers. Automated High-Throughput Liquid Chromatography A high-throughput capillary HPLC system has been successfully designed and developed in-house and is now being used for routine analysis of proteomic samples. This system is based on doubling the fluidic components of a design previously developed in our laboratory which allows it to overlap the processing of four separation columns by applying a novel technique for interfacing the columns with a mass spectrometer based on encoding translation stages. The 4-column system has a number of advantages over the original 1-column design developed in our laboratory including throughput, cost, and size. First, the 4-column system has a duty cycle of ~100% with no accompanying loss in data quality compared to the 1-column system. In 24 hours the 4-column system has a throughput of ~14 samples compared to only ~7 samples for the 1-column system. Second, this increase in sample throughput can be accomplished by upgrading an existing 1-column system at a substantial savings compared to the alternative of acquiring another 1-column system and a mass spectrometer. Finally, upgrading an existing system does not increase the footprint of the LC/MS system(s) needed to achieve the increase in throughput while acquiring a new system doubles it. Automated Sample Handling Development and refinement of protocols that can be performed on a Beckman Biomek FX automated liquid-handling robot are in progress. The NCRR collaborative project with Desmond Smith has contributed significantly to the development and refinement of automated sample handling due to the extensive numbers of small samples associated with this project - pieces of mouse brain tissue referred to as brain voxels. A single mouse brain can yield ~400-600 1 l voxels and methods are being developed to process them in 96-well plates. The proteins that are extracted from each voxel are tryptically digested using a protocol developed for the Biomek FX for processing proteome samples in solution (i.e., not isolated in a 2D-PAGE gel). Future work on this project will involve automating more steps of the brain voxel sample-handling protocol in addition to transferring samples ready for MS analysis to an LC-MS system in a 96-well plate format (currently the LC-MS systems are configured only for individual sample vials). Planar FAIMS Development at PNNL Having previously reported the development of software for a priori simulation of Field Asymmetric waveform Ion Mobility Spectrometry (FAIMS) analyzers, in 2005 we used the package to optimize FAIMS performance as a function of gap curvature, in particular, comparing planar (p-) and cylindrical (c-) geometries. Calculations revealed that p-FAIMS offers the maximum resolution, exceeding that of c-FAIMS with typical curvatures by ~2 - 5 times. The improvement applies to ions of any type and increases at higher absolute compensation voltages (CV). Guided by modeling, we have built and evaluated a new high-resolution p-FAIMS unit. In agreement with simulations, the resolution gain is up to 300%, increasing with increasing CV. That performance has enabled difficult isomeric separations not possible by FAIMS previously, such as distinguishing (i) protonated Leucine and Isoleucine and (ii) at least four H+bradykinin conformers vs. two found using c-FAIMS. Separation of protein conformers has also improved, though by a smaller margin (likely because of inherent diversity of conformer ensembles). The new p-FAIMS also features an integrated ESI interface providing effective ion desolvation, making the system suitable for effective analysis of proteomic and other biological samples. Simulations also show the superiority of p- over c- FAIMS in terms of resolution/ sensitivity balance, meaning a higher resolution at equal sensitivity or higher sensitivity at equal resolution. However, broad adoption of p-FAIMS was impeded by lack of effective MS interfaces. We have developed and demonstrated a new slit-aperture MS (and IMS) interface that, in conjunction with an electrodynamic ion funnel, provides a better capture of diffuse ribbon-shaped ion beams exiting p-FAIMS and thus improves the sensitivity of FAIMS/MS analyses. Efforts to increase the ion utilization at the p-FAIMS/MS junction further are in progress.
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