Mass spectrometry has become a method of choice for identifying and characterizing small quantities of proteins in complex mixtures. However, the ability to perform the identification in a high- throughput fashion has depended on the availabilit of protein sequence databases. This means that proteins from organisms with unsequenced genomes (e.g. peptide toxins) and proteins that modify their primary sequence rapidly in response to the environment (e.g. antibodies) have been excluded from high-throughput analysis. We propose to develop algorithms and software along with improving laboratory methods that make sequencing of antibodies and peptide toxins a fast and low-cost effort. This will allow us to access the circulating antibody repertoire of individuals for clinical application including vaccine development, and to access the vast number of bioactive venom components for basic research and ion-channel drug development. For the laboratory improvements, antibody peptides and toxins will be chemically labeled to improve spectral quality and we will use different types of mass spectrometric fragmentation. Data acquisition will be optimized to facilitate identification of diagnostically relevant peptides and a gas- phase digestion strategy will be used to increase the sequence coverage for larger peptides. We propose to develop improved algorithms for sequencing of antibodies and peptide toxins. These will integrate de novo and database sequencing and will include candidate generation algorithms incorporating multiple channels of information: spectra from different charge states and fragmentation methods, homology constraints, composition constraints, and in silico mutation of databases. Improved scoring algorithms will also be developed using subtle spectrum clues, currently used only in manual de novo sequencing. We will produce prototype software, and benchmark it against manually annotated mass spectra. The software will then be applied to automatically sequence a large set of antibody data from long- term non-progressors of HIV, and spider and cone snail toxin data.
We propose to develop algorithms and software that make sequencing of antibodies and peptide toxins a fast and low-cost effort. This will enable the effortless generation of large amounts of antibody and peptide toxin sequences;a critical step for vaccine and ion-channel drug development, respectively.
| Cordeiro, Francielle Almeida; Coutinho, Bárbara Marques; Wiezel, Gisele Adriano et al. (2018) Purification and enzymatic characterization of a novel metalloprotease from Lachesis muta rhombeata snake venom. J Venom Anim Toxins Incl Trop Dis 24:32 |
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| Marino, Fabio; Bern, Marshall; Mommen, Geert P M et al. (2015) Extended O-GlcNAc on HLA Class-I-Bound Peptides. J Am Chem Soc 137:10922-10925 |
| Aman, Joseph W; Imperial, Julita S; Ueberheide, Beatrix et al. (2015) Insights into the origins of fish hunting in venomous cone snails from studies of Conus tessulatus. Proc Natl Acad Sci U S A 112:5087-92 |
| Safavi-Hemami, Helena; Gajewiak, Joanna; Karanth, Santhosh et al. (2015) Specialized insulin is used for chemical warfare by fish-hunting cone snails. Proc Natl Acad Sci U S A 112:1743-8 |
| Wiezel, Gisele A; dos Santos, Patty K; Cordeiro, Francielle A et al. (2015) Identification of hyaluronidase and phospholipase B in Lachesis muta rhombeata venom. Toxicon 107:359-68 |
| Li, Yinyin; Cross, Frederick R; Chait, Brian T (2014) Method for identifying phosphorylated substrates of specific cyclin/cyclin-dependent kinase complexes. Proc Natl Acad Sci U S A 111:11323-8 |
| Gajewiak, Joanna; Azam, Layla; Imperial, Julita et al. (2014) A disulfide tether stabilizes the block of sodium channels by the conotoxin ?O§-GVIIJ. Proc Natl Acad Sci U S A 111:2758-63 |
| Liu, Suli; Im, Hogune; Bairoch, Amos et al. (2013) A chromosome-centric human proteome project (C-HPP) to characterize the sets of proteins encoded in chromosome 17. J Proteome Res 12:45-57 |
| Muellenbeck, Matthias F; Ueberheide, Beatrix; Amulic, Borko et al. (2013) Atypical and classical memory B cells produce Plasmodium falciparum neutralizing antibodies. J Exp Med 210:389-99 |
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