Protein-Peptide Sequencing

Sequence Variations in Interlobe Connecting Peptide Protein Sequence"... 

Linear peptides are the most common example of peptide chimeras. In this strategy two different peptide/protein sequences are tandemly linked. These compounds can be chemically assembled using stepwise (16,17) or convergent solid phase synthetic procedures (18). The length and/or the amino acid composition of desired sequences may dictate different approaches. The other possibility is the attachment of monomeric peptides by the formation of nonpeptide... 

An off-line approach that is simple and useful for peptide/protein sequencing using 5-10 picomoles of material has been demonstrated. Peptide and protein samples were first separated by capillary electrophoresis. Selected peaks were fraction collected and analyzed by both nano-electrospray mass spectrometry and Edman sequencing. A standard peptide mixture, a tryptic-digested protein and intact proteins were used to illustrate this method. Successful fraction collection of each component required reproducible electropherograms, the ability to automatically switch the outlet buffer vessel and the ability to maintain electrophoretic integrity while eluting a peak of interest into a small outlet buffer... 

The ECD process, by its nature, is a very rapid process and bond dissociation occurs faster than the redistribution of intramolecular vibrational energy that occurs with CID. This explains the dissociation of the strong N-C, amine bonds in the presence of the weaker C—N amide bonds in peptides and proteins.93,94 Consequently, any labile PTMs (e.g., phosphorylation, sulfation, 7-carboxylation, N- and O-glycosylation) are preserved and may be unequivocally located in the peptide/protein sequence. See also discussion in Section 9.10.3.2.6 on the use of ECD/ETD and CID/IRMPD for protein/peptide sequencing, and Table 4. 

Solid-phase sequencer, an apparatus for peptide/protein sequence analysis. Automated primary structure determination of peptides and proteins using the solid-phase sequencing method is based on cova-... 

Partial amino acid (aa) sequences of the most prevalent extract proteins were determined using an Applied Biosystems 470A Gas Phase Peptide/Protein Sequencer coupled to a 120A HPLC and DEC computer in the Protein Chemistry Core Facility (PCCF) of the University of Louisville. The aa compositions of major protein isoforms were determined using a Waters Pico-Tag System in the PCCF. 

Although BIRD is not ideal for peptide/protein sequencing, the fact that the temperature of the ions environment can be measnred accurately means it is eminently suitable for determining rate constants, activation energies, and dissociation energies. 

Mote recently, ETD, a dissociation technique very similar to ECD, was introduced for peptide/protein sequencing [56-58]. In ETD, multiply-chaiged cations react with an anion radical (A, e.g., SO2, SFg", anthracene, fluoranthene), leading to electron transfer and the generation of both odd- and even-electron fragments and molecular species with lower charge states as follows ... 

Specific peptide/protein sequences are required to obtain targeted functionalities. The method of choice for peptide synthesis of more than one amino acid type depends largely on the length of the sequence. Short peptide sequences, typically less than 50 residues in length, are usually prepared by SPPS, shown in Figure 7(b)This method allows for the production of complex sequences, as seen in natural proteins, and the incorporation of nonnatural amino acids. 

ChemSketch has some special-purpose building functions. The peptide builder creates a line structure from the protein sequence defined with the typical three-letter abbreviations. The carbohydrate builder creates a structure from a text string description of the molecule. The nucleic acid builder creates a structure from the typical one-letter abbreviations. There is a function to clean up the shape of the structure (i.e., make bond lengths equivalent). There is also a three-dimensional optimization routine, which uses a proprietary modification of the CHARMM force field. It is possible to set the molecule line drawing mode to obey the conventions of several different publishers. 

The amount of sample required is quite small as little as 10 mole is typical So many peptides and proteins have been sequenced now that it is impossible to give an accurate count What was Nobel Prize winning work m 1958 is routine today Nor has the story ended Sequencing of nucleic acids has advanced so dramatically that it is possible to clone the gene that codes for a particular protein sequence its DNA and deduce the structure of the protein from the nucleotide sequence of the DNA We 11 have more to say about DNA sequencing m the next chapter... 

The importance of linked scanning of metastable ions or of ions formed by induced decomposition is discussed in this chapter and in Chapter 34. Briefly, linked scanning provides information on which ions give which others in a normal mass spectrum. With this sort of information, it becomes possible to examine a complex mixture of substances without prior separation of its components. It is possible to look highly specifically for trace components in mixtures under circumstances in which other techniques could not succeed. Finally, it is possible to gain information on the molecular structures of unknown compounds, as in peptide and protein sequencing (see Chapter 40). 

The "overlap" information allows the original protein sequence to be generated. In this example, the sequence D-E-F-G in the fourth peptide proves that the first and second peptides must have been joined originaliy at E,F and not A,H. 

Protein sequencing by creation of overlap peptides (mapping),... 

As described earlier, translation of the EPSPS mRNA of plants results in the formation of a protein which has an AJ-terminal extension. The AJ-terminal extension, referred to as the chloroplast transit peptide, is necessary and sufficient for the import of the preprotein by the chloroplast. Once imported by the chloroplast, the transit peptide is cleaved releasing the mature enzyme. As expected, introduction of the EPSPS transit peptide to other protein sequences results in the importation of the fusion protein by the chloroplast. 

H Taketomi, Y Ueda, N Go. Studies on protein folding, unfolding and fluctuations by computer simulation. 1. The effect of specific ammo acid sequence represented by specific mter-umt interactions. Int J Peptide Protein Res 7 445-459, 1975. 

The general idea of peptide sequencing by Edman degradation is to cleave one amino acid at a time from an end of the peptide chain. That terminal amino acid is then separated and identified, and the cleavage reactions are repeated on the chain-shortened peptide until the entire peptide sequence is known. Automated protein sequencers are available that allow as many as 50 repetitive sequencing cycles to be carried out before a buildup of unwanted by products interferes with the results. So efficient are these instruments that sequence information can be obtained from as little as 1 to 5 picomoles of sample—less than 0.1 /xg. 

Overexpression of apoaequorin (Inouye et al., 1989, 1991). To produce a large quantity of apoaequorin, an apoaequorin expression plasmid piP-HE containing the signal peptide coding sequence of the outer membrane protein A (ompA) of E. coli (Fig. 4.1.12) was constructed and expressed in E. coli. The expressed apoaequorin was secreted into the periplasmic space of bacterial cells and culture medium. The cleaving of ompA took place during secretion thus the... 

Besides sensitive methods for the analysis of proteins, bioinformatics is one of the key components of proteome research. This includes software to monitor and quantify the separation of complex samples, e.g., to analyze 2DE images. Web-based database search engines are available to compare experimentally measured peptide masses or sequence ions of protein digests with theoretical values of peptides derived from protein sequences. Websites for database searching with mass spectrometric data may be found at http //www.expasy.ch/tools, http //prospector.ucsf. edu/ and http //www.matrixscience.com. 

THE EDMAN REACTION ENABLES PEPTIDES PROTEINS TO BE SEQUENCED... 

Meisel, H. and FitzGerald, R. J. (2000). Opioid peptides encrypted in intact milk protein sequences. Br. J. Nutr. 84, S27-S31. 

Montecucchi PC, de Castiglione R, Piani S, Gozzini L, Erspamer V. Amino acid composition and sequence of dermorphin, a novel opiate-like peptide from the skin of Phyllomedusa sauvagei. Int J Peptide Protein Res 1981 17 275-283. 

Marsden BJ, Nguyen TM-D, Schiller PW. Spontaneous degradation via diketopiperazine formation of peptides containing a tetrahydroisoquinoline-3-carboxylic acid residue in the 2-position of the peptide sequence. Int J Peptide Protein Res 1993 41 313-316. 

Henzel, W. J., Billeci, T. M., Stults, J. T., and Wong, S. C. (1993). Identifying proteins from two-dimensional gels by molecular mass searching of peptide fragments in protein sequence databases. Proc. Natl. Acad. Sci. USA 90, 5011-5015. 

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