The objective of this proposal is to introduce a product that will enable protein separations by gel electrophoresis (SDS-PAGE) to be analyzed as peptides by mass spectrometry in an efficient, high-throughput manner. Such a device will offer investigators in biology and medicine a compelling new method for analyzing complex proteomic samples (100s - 1000s of individual components) by directly addressing the most persistent challenges in proteomic research;how to manage sample component diversity, in both number and concentration, to maximize sample species detection while preserving information pertinent to the biology of the system. Availability of the product resulting from this work will address these challenges by coupling the most efficient, highest resolution, protein separation technique with the accuracy, sensitivity and speed of mass spectrometry detection of peptides in a manner that is thorough. Presently, this link does not exist. Subtle details in the variants of a protein or protein family are implicated in the detection or propagation of menacing diseases like cancer, alzheimier's, diabetes and arthritis. Therefore, experimental protocols that enable the discernment and identification of these features are necessary to meet research objectives. This new product will offer an attractive experimental scheme to proteomic research efforts because sample separation will be performed at the level of intact proteins, preserving valuable information regarding polymorphisms and post- translational modifications within given species. The goal of this phase 1 study is to demonstrate a system for coupling protein gel separations with mass spectrometry detection of peptides that is simple, efficient, sensitive, reproducible and easily implemented.
Four specific aims for this phase 1, feasibility study are listed with follow-up discussion on how they will be tested and evaluated for success. Since its introduction by Laemilli ~ 40 years ago, SDS-PAGE has remained one of the most popular methods for separating and analyzing proteins and protein mixtures. The near universal applicability, resolving power, consistency and simplicity of SDS-PAGE protein separation remains unmatched. Over the past ~15 years, the mass spectrometer has emerged as the detector of choice in experimental proteomics. The reasons for this include the critical, analytical-traits of accuracy, sensitivity and speed in detection of peptides and the ability to selectively fragment peptides to obtain primary sequence information enabling protein identification. Unfortunately, currently, there is no efficient and thorough transition between gel separation of proteins and mass spectrometry of peptides. Current protocol that attempts to link these two powerful experimental methods is largely manual, time consuming and often incomplete and imprecise. Outlined and preliminarily demonstrated in this proposal is a novel membrane that has the ability to both capture and digest protein(s) orthogonally blotted from SDS-PAGE gels. Specific selection and composition of the chemistry built into the membrane acts to process protein(s) in-place, thoroughly digesting them into their constituent peptides while retaining and limiting diffusion, until their deliberate release by application of the appropriate elution solvent. It is believed that the duel tasks performed by this membrane can take place in a timely and precise manner making it an extremely viable commercial product. What is needed to complete the system, as envisioned, is a means for en-masse elution of bound peptides from membrane so that separations encompassing entire gels can be processed at once. The concept for this procedure is the creation of elution plates that will allow elution solvent to pass through the membranes under the force of centrifugation. The design of these elution plates will allow the elution solvent and analyte to be easily collected with the resolution of separation preserved. The goals for this product are these. 1) To harness the near universal applicability, convenience, and resolving power of gel-based protein separations to separate and preserve the primary structure of complex protein mixtures. 2) To harness the efficiency and convenience of electrophoresis blotting to thoroughly remove all proteins from the resolving gel without the need of detection. 3) To thoroughly digest the gel-separated proteins into their constitute peptides while maintaining the resolution of separation. 4) To capture and concentrate the resulting peptides and elute them in a liquid medium optimized for detection by mass spectrometry. 5) Use the accuracy, sensitivity and speed of mass spectrometry detection of peptides to interrogate the sample to fulfill the objectives of the research. The key components of the product will be the uniquely designed bi-functional membrane and the novel elution plate that will enable the en-masse elution of the enzyme-digested proteins (peptides) in a solution compatible with detection by mass spectrometry.
Contingents of the imminent diseases cancer, Alzheimier's, diabetes and arthritis, all have variants of particular protein(s) implicated in their detection or propagation. Novel experimental protocols that aid in the discernment and identification of these often, subtle details are necessary to meet the objectives of efforts to diagnose, understand and treat these diseases. Outlined in this proposal is a product that will provide a link between two o most powerful technologies available to researchers in biology and medicine where currently one does not exist. The goal of this proposal is to demonstrate the means for coupling these technologies in a way that is simple, efficient, sensitive, reproducible and easily implemented.