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May 15, 2013 (Vol. 33, No. 10)

Mass Spec Provides Peek into PTMs

  • “We want to conduct our investigations using a broad as possible approach and dig deep into any direction that we could go. There is so much variation across different tissues, as well as modifications in peptides, that this gap between biological PTMs and spectral libraries is widening with massive efforts in both realms,” he explains when describing the many advantages of searching mass spec data against spectral libraries (collections of reference spectra) instead of using the more traditional database search approach.

    Dr. Bandeira is currently developing an even larger compendium of proteomics mass spec data that will house PTM data from different issues. “This MassIVE (Mass spectrometry Interactive Virtual Environment) community-wide repository we are developing will serve as a resource to converge all existing information on PTMs and mass spec data, including data from the latest types of mass spec instruments. Current databases provide limited information in that generally, these do not offer raw data associated with a mass spec report or vice versa.”

    Dr. Bandeira has also expressed his hopes that researchers in the field of proteomics engage in data sharing. “Our vision in setting up this MassIVE repository at UCSD is that researchers will be able to easily access, upload their data, and even search what is available at the site. Even in its current version, our ProteoSAFe system has already enabled the analysis of over 1 billion spectra from over 3,000 users. We are aware of how important spectral data is with regard to PTMs and thus putting all this information together in one site, with users having free access to the information, will definitely enhance the analysis and understanding of PTMs,” says Dr. Bandeira.

    For Marshall Bern, Ph.D., vp of Protein Metrics, screening for PTMs can be very challenging because it highly relies on prior knowledge and existing databases. “The current list of reported PTMs is now larger compared to previous years, and this is due to the rapid improvement in analytical tools in proteomics. Although this may be a major advancement in the field, this also increases the need for a reliable and rapid algorithm that understands what peptides one is trying to identify.

    “With the knowledge that each amino acid occurs at multiple sites along a polypeptide chain, multiplied by the modification that marks each amino and changes its mass state, then you end up with a larger number of protein possibilities to search in a database. Since PTMs are generally characterized by mass states, if you have a longer peptide, then you will most probably have more mass states per amino acid, thus making it harder to search through a comprehensive protein resource,” says Dr. Bern.

    Protein Metrics reports that it offers one of the best solutions in searching databases for protein analysis. Dr. Bern explains that their new proteomics search engine, Byonic, addresses the most common weaknesses in searching databases.

    “Byonic offers a wildcard search that assists in finding matches, including specific changes within a peptide such as a PTM on the N-terminus of a chain,” says Dr. Bern. He also explained that although this approach may be slower than known-modification searches, it decreases the chance of missing a PTM by considering every possible mass shift within a range.

    “The software also helps in finding matches that carry more than two modifications. Byonic can identify hundreds of times more PTMs than any existing software including N- and O-linked glycans. Byonic lets the user ask and answer more questions since it covers more types of PTMs,” he discusses.

  • Proteolytic PTMs

    Christopher M. Overall, Ph.D., Canada research chair in metalloproteinase proteomics and systems biology at the Centre for Blood Research, University of British Columbia, has investigated proteolytic PTMs extensively.

    “Every protein in a cell has been subjected to the activities of proteolytic proteins or proteases, which are highly ubiquitous and comprise the second-largest enzyme family in man. Proteins that undergo cleavage often change their biological function. For example, chemokines, which are chemoattractant cytokines, undergo modifications that change their activity and binding capacity, influencing their role in inflammation and immunity,” says Dr. Overall.

    He has also explained that matrix metalloproteinases act on these chemokines, which can render these as an agonist or antagonist to a cell by switching its role as a receptor and enhancing the influx of specific ions into particular cells. In other cases, cleavage of chemokines may even result in its disengagement from its matrix, he adds.

    Dr. Overall’s research team was the first to use the yeast two-hybrid screen for protease substrate finding, utilizing inactive mutants of proteases and isolated domains of proteases as bait.

    “While effective, it is slow. The field of degradomics, or protease substrate discovery, requires the newest techniques in finding substrates of proteases. Our group has developed highly novel proteomic approaches to specifically identify cleaved proteins in tissues. We are currently funded by the Canadian Institutes of Health Research and the Natural Sciences and Engineering Research Council to embark on an extensive effort to develop new polymers for proteomics by using high-throughput selection of modified peptides,” says Dr. Overall.

  • Top-Down Approach

    Click Image To Enlarge +
    A top-down approach to MS-based proteomics using histone H4: The identification of PTMs in a natural form allows precise identification of proteins without the disruptive effects of protein digestion that is commonly employed in the classical bottom-up approach. [Northwestern University]

    One fresh and interesting strategy for the study of PTMs is the top-down proteomics approach, which involves the screening of whole proteins at the level of their primary structure.

    Proteins are highly dynamic molecules that are in many ways more difficult to analyze than the genome, according to Neil L. Kelleher, Ph.D., professor in molecular biosciences, chemistry, and the Feinberg School of Medicine at Northwestern University. Furthermore, use of the classical bottom-up proteomics approach of digesting peptides using proteases and subjecting these fragments to LC/MS does not maximize the information that could be gathered.

    “The top-down proteomics approach does not involve protein digestion and thus, identification is mainly derived from fragmentation of the intact polypeptide. This major leap in technology development in proteomics will significantly enrich protein identification and characterization efforts,” Dr. Kelleher notes.

    “Capturing proteins in their near-natural form not only increases the information content obtained, but may also prevent further confusion with regard to protein terms such as isoforms, variants, and species,” he explains. Dr. Kelleher and a new Consortium for Top Down Proteomics have proposed the use of the term “proteoform” to describe various proteins products generated from a single gene, including those derived from alternative splicing, genetic variations, and PTMs.

    “The many previous terms did not describe cleanly the wide range of protein forms that have been discovered and reported, which created some degree of confusion in protein nomenclature. It is apparent that grouping proteins based on their gene origin allows a more direct and informative approach to protein identification in a gene-specific fashion,” he adds.

    Dr. Kelleher has articulated a new “cell-based” version of the Human Proteome Project, which would call for defining proteomes based on specific human cell types.

    “The plan is to detect and characterize a billion proteoforms emanating from the ~20,000 genes as expressed and processed in the diverse cell types in the human body. Both bottom-up and top-down approaches can be utilized together to address questions in proteomics and human disease with greater precision,” he emphasizes.


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