Bottom-up proteomics is a technique where proteins from a sample, including complex human fluids, and tissue and cell samples, are digested enzymatically into its constituent peptides before analytical characterization is performed using liquid chromatography-tandem mass spectrometry. From these peptide fragments, the protein mixture contained in the original sample are reassembled, identified, and quantified (if desired). It is the predominant method used for protein analysis, and it is used broadly to uncover genetic expression of proteins and their variants in a system of interest, including differential expression to determine for the presence of disease and for the efficacy of disease treatment. There are many steps between the initial protein isolation and injection onto the liquid chromatograph for mass spectrometry analysis. The goal, of course, is that the final assembled proteins reflect accurately the proteins contained in the original sample. The weakest step appears to be the final purification, which uses a reverse-phase adsorbent. Recoveries here for a reference sample are only about 50%, and this is reflected in the sequence coverage of the reassembled proteins. Here we propose an alternative based on an unconventional electrophoresis method. It offers a much more complete peptide recovery that has the potential to substantially eliminate peptide bias. The new method offers a means to concentrate by at least 30-fold while removing impurities such as salts, denaturants such as urea, reducing agents such as dithithreitol, and most surfactants. If successful, this peptide recovery method should enable more complete and useful protein characterization of a sample of interest.

Public Health Relevance

Bottom-up proteomics is an analytical technique that is the cornerstone of proteomics research. Proteins and their variants have implications for disease discovery and monitoring its progression, for disease diagnosis, and for the efficacy of companion diagnostics. It relies on many intermediate steps between sample procurement from whatever source to convert it to a form suitable for characterization by liquid chromatography/electrospray tandem mass spectrometry. We have identified the weakest link in those steps ? the final peptide purification and isolation ? and propose to develop a powerful alternative method to recover a sample that will reflect more accurately the proteins, protein fragments and variants the initial sample contains. We believe that it will have a profound effect on determining disease and its progression by providing protein data that is of much higher quality and without the bias that often is present using existing methods. This, in turn, will enable improved fundamental disease understanding, and facilitate improved disease diagnostics and development of therapeutics against those diseases.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Small Business Innovation Research Grants (SBIR) - Phase I (R43)
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Special Emphasis Panel (ZRG1)
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Krepkiy, Dmitriy
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Sonata Biosciences, Inc.
United States
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