De novo protein sequencing

The major steps in de novo protein sequencing are (1) digest multiple samples of the unknown with several enzymes to produce multiple sets of peptides, (2) analyze these sets by MS to determine their miz ratios, (3) use MS/MS to obtain the amino acid sequence of each peptide, and (4) cross-reference the sequenced chains, cleaved by the different enzymes, to derive the overall sequence of the protein (Fignre 3.31). 

Analysis of the intact protein provides molecular mass. 

Not all peptides are observed, nor are they fully sequenced, from the data acquired using proteolytic enzymes 1 and 2. Complementary sections of amino acid sequences from the two enzymes are aligned to provide full coverage. 

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In comparison with the bottom-up approach, the top-down approach for de novo protein sequencing is faster and can be applied on proteins in mixture. However, for analysis of post-translational modifications, these two approaches are complementary and a combination of them is interesting. The bottom-up approach allows detection of low-stoichiometric modifications even if a high-stoichiometric modification is missed. Furthermore, enzymatic digestion leads to the loss of all connectivity information from the original protein species. On the other hand, the top-down approach allows observation of the global pattern of... 

Mass spectrometry-based protein identification protocols have propelled proteomics to the forefront of biomedical research. Provided with available genomic and/or protein sequence information in databases, protein identification by MALDl-based peptide mass fingerprint mapping and LC-MS/MS and MALDl-TOF/TOF peptide sequencing with CAD fragmentation is efficient. However, in situations in which sufficient genome and protein sequence data are unavailable, mass spectrometric methods can be used for de novo protein sequencing. The mass measurement accuracy of MS provides a unique capability to... 

Dahiyat, B.I., Mayo, S.L. De novo protein design fully automated sequence selection. Science 278 82-87,... 

Johnson R.S. and Taylor J.A. (2000), Searching sequence databases via de novo peptide sequencing by tandem mass spectrometry, in Methods in Molecular Biology, Vol. 146, Mass Spectrometry of Proteins and Peptides, pp. 41-61, Chapman J.R., Ed., Humana Press, Totowa, NJ. 

G. Cagney, A. Emili, De novo peptide sequencing and quantitative profiling of complex protein mixtures using MCAT, Nat. BiotechnoL, 20 (2002) 163. 

Serotonin induces secretion of IL-16 from CDS but not from CD4 T cells. This induction does not require de novo protein synthesis. Serotonin, via serotonin type 2 receptors, may promote the recruitment of CD4 T lymphocytes into an inflammatory focus. IL-16 is released in response to an antigen, mitogen, histamine, or serotonin. Histamine induces secretion of IL-16 by interaction with H2-type histamine receptors on CDS cells, and serotonin induces secretion by interaction with S2-type receptors. The mechanism of IL-16 secretion is unknown however, the intracellular 80-kDa molecule suggests the necessity of protein processing, resembHng IL-1 (which also lacks leader sequences) that is secreted after processing and formation of pore complexes. ... 

The amino acid sequence determination of peptides through de novo peptide sequencing procedure is one of the most familiar applications of mass spectrometry. A precise knowledge of the amino acid sequence of peptides is required in many situations - to understand their biological functions, to characterize components of the metabolic cycle of precursor proteins, to map changes in the metabolic profile of a peptide family caused by... 

B. I. Dahiyat, C. A. Sarisky, S. L. Mayo. De novo protein design towards fully automated sequence selection. J Mol Biol. 1997, 273, 789-796. 

Dahiyat, B. I., and Mayo, S. L. (1997). De novo protein design Fully automated sequence selection. Science 278(5335), 82-87. 

The molecular middle ground between nonheme di-iron enzymes and their small molecule synthetic analogues has been explored by de novo protein design. A protein can be designed to fold into a predetermined tertiary structure without patterning the sequence after any natural... 

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