Mass Spectrometry Imaging of Protein Gels

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DESCRIPTION (provided by applicant): We propose to develop a high-throughput, integrated top-down mass spectrometry imaging platform to identify and characterize protein isoforms and expression profiles from complex biological samples and to elucidate effects of protein modifications on protein function and interactions. The technology combines the premier, high resolution capabilities of gel electrophoresis with the sensitivity and high mass accuracy of mass spectrometry (MS). Intact proteins separated by isoelectric focusing gel electrophoresis will be imaged from immobilized pH gradient (IPG) polyacrylamide gels (the first dimension of classic 2D-PAGE) by automated, high-speed matrix-assisted laser desorption/ionization (MALDI) MS. This will be followed by gel-wide chemical and enzymatic methods and further interrogation by MALDI-MS/MS to provide information on sequence, identification, and post-translational/transcriptional modifications. Our MS imaging approach is distinguished from other top-down proteomic and mass spectrometry methods in that (1) we seek to track and to elucidate modified proteins from separation formats employed by biologists to monitor presence, amount, enzymatic activity, and interactions of discrete protein isoforms, (2) we seek to profile intact proteins from complex samples that are available in only sub-microgram quantities, and (3) we seek to extend intact protein analyses to membrane proteins and small open reading frame products. The "Virtual 2D gel/MS" platform may potentially lead to mass spectrometric visualization of ultrahigh resolution separations in less than 30 minutes, linking the benefits of "top-down" and "bottom-up" proteomics. Our vision is that the technology will provide biologists and mass spectrometrists with nearly unprecedented capability to link dynamic protein modifications to specific disease or cellular responses, and that the platform can be widely deployed. PUBLIC HEALTH RELEVANCE: We propose to develop a high-throughput technology to profile protein status, including modifications, as well as membrane proteins and small molecular mass proteins. Current methods have limitations that generally prevent biologists from accessing complete information about protein forms; this technology would alert them to absent, but necessary information. More detailed information about protein forms and how they could be altered after injury or by disease can be obtained.