Method to Map Protein Landscapes
Inactive Publication Date: 2018-11-29
WISCONSIN ALUMNI RES FOUND
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Benefits of technology
The present invention provides methods and systems for analyzing polypeptides with increased sequence coverage and improved analysis of protein expression and modification. This is achieved by using multiple proteases or chemical agents to digest the polypeptides and using deep sequencing technology to generate comprehensive mass spectrometry data on each polypeptide. The methods also involve digesting multiple samples of the polypeptide with different proteases or chemical agents to increase the accuracy of the analysis. The invention also provides a way to compare the polypeptides and identify differences in amino acid abundance between them. Overall, the invention improves the accuracy and efficiency of analyzing polypeptides and their modification patterns.
Problems solved by technology
Trypsin's moderate sequence coverage is sufficient for most proteomics experiments but does not provide sufficient sequence coverage for distinguishing various forms of a protein (proteoforms).
However, because the entire protein sequence is not detected, it is often impossible to determine whether the expressed protein is present in a modified, spliced, or truncated form.
As such, this level of information is often not collected during conventional proteomic analyses, and there is currently no good way to monitor which form(s) of a protein exists in the cell, or which form(s) of a protein are present in a purified protein drug therapeutic.
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[0040]In a first example, a large scale study of the yeast proteome was performed. The proteome was digested with the following six proteases: trypsin, Lys-N, Lys-C, Glu-C, chymotrypsin, and Asp-N. Over 6,000 proteins (95% of the yeast proteome) were identified with a mean sequence coverage of 80%. To enable the identification of endogenous peptides produced by natural proteolytic activity in the cell, the data was processed with a no enzyme search. Of special interest was the mitochondrial proteome, most of which is subject to N-terminal processing of the full protein sequence. The well characterized coenzyme Q5 (COQ5) protein, a methyltransferase, essential to the ubiquinone biosynthesis pathway, was selected. COQ5 undergoes post-translational processing by N-terminal truncation. FIG. 1 illustrates the sequencing depth of COQ5, with over 250 quantified peptides.
[0041]The peptide mapping reveals the dynamic range of peptides from multiple proteases, but also putative proteoforms. T...
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In shotgun proteomics, generally only a fraction of peptides from a parent protein are actually detected. Because a large portion of the protein sequence is not detected, it is often impossible to determine whether the expressed protein is present in a modified, spliced, or truncated form. Provided herein are methods and systems for analyzing polypeptides which allow for the increase of the mean sequence coverage of a protein concomitant with bioinformatics analysis in order to distinguish putative proteoforms with improved amino acid resolution. Aspects of the invention include (1) a deep sequencing strategy to provide more protein sequence coverage than is typically achieved, and (2) a computational approach to view protein expression across its full length and identify regions of the protein that are potentially subject to such regulation. This technology has global utility in proteomics and will be of particular use for the analysis of biosimilar protein drug therapeutics.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application claims priority from United States Provisional Patent Application No. 62 / 511,011, filed May 25, 2017, which is incorporated by reference herein to the extent that there is no inconsistency with the present disclosure.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT[0002]This invention was made with government support under GM 118110 and GM108538 awarded by the National Institutes of Health. The government has certain rights in the invention.BACKGROUND OF THE INVENTION[0003]Mass-spectrometry-based proteomics is a key technology for studying the proteome, which can comprise canonical gene products, alternative gene products, post-translational modifications (PTMs), non-synonymous single nucleotide polymorphisms (SNPs) and other sequence variations. The most prevalent paradigms are top-down proteomics and bottom-up proteomics, also known as shotgun proteomics. In shotgun proteomics, proteins in a sample underg...
Claims
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Login to View More IPC IPC(8): G01N33/68G06F19/24G06F19/22H01J49/00G16B30/20G16B40/10
CPCG01N33/6848G06F19/24G01N33/6824G06F19/22G01N33/6842H01J49/0036H01J49/004G01N2570/00G16B40/30G16B40/10G16B30/20G16B30/00G16B40/00
InventorCOON, JOSHUAMARX, HARALD
OwnerWISCONSIN ALUMNI RES FOUND