Sequencing a Peptide

Efficiency in Peptide Sequencing A peptide with the primary structure Lys-Arg-Pro-Leu-Ile-Asp-Gly-Ala is sequenced by the Edman procedure. If each Edman cycle is 96% efficient, what percentage of the amino acids liberated in the fourth cycle will be leucine Do the calculation a second time, but assume a 99% efficiency for each cycle. 

A powerful method of sequencing a peptide from the TV-terminal end is the Edman degradation in which phenyl isothiocyanate, C6HsN=C=S, reacts selectively with the terminal amino acid under mildly basic conditions. If the reaction mixture is then acidified, the terminal amino acid is cleaved from the peptide as a cyclic thiohydantoin, 8 ... 

In theory, Edman degradations could sequence a peptide of any length. In practice, however, the repeated cycles of degradation cause some internal hydrolysis of the peptide, with loss of sample and accumulation of by-products. After about 30 cycles of degradation, further accurate analysis becomes impossible. A small peptide such as bradykinin can be completely determined by Edman degradation, but larger proteins must be broken into smaller fragments (Section 24-9E) before they can be completely sequenced. 

Most small peptides derived from protein sources contain a variety of polar and non-polar amino acids and as such are more difficult to handle. Nevertheless appreciation of the problems and considered chemical manipulation have allowed their MS elucidation [177-184] although a practical limit of about six amino acids in the peptide is reached before degradative procedures become advisable. An important step before sequencing a peptide by mass spectrometry is whenever possible to obtain the amino acid content by hydrolysis and conventional column chromatographic analysis. This assists in the selection of any chemical pretreatment and with the spectral interpretation. A variety of small peptides has been identified by a combination of methods and include 5 - 0X0 - L - prolyl - L - histadyl - L - prolinamide (2-pyrollidone-5-carboxylyl-... 

Sequencing a peptide by the recording of a single spectrum is a very attractive proposition which stimulated considerable research. The classical process of mass spectrometry requires a volatile sample which when injected into an evacuated chamber evaporates. The vapors are then exposed to a beam of electrons. Under electron bombardment the molecular ion and fragment ions are produced. For this purpose most peptides are not sufficiently volatile. 

Rgure 1. Scenarios depicting the possible solvation or aggregation states of a polymer-bound peptide sequence, (a) Peptide and polymer chains (bold) are fully solvated, (b) Peptide chains are intramolecularly aggregated whilst polymer is solvated, (c) Peptide chains are well solvated whilst the polymer backbone is poorly solvated, (d) Peptide chains are intermolecularly aggregated whilst polymer is solvated, effectively increasing the overall level of cross-linking in the matrix. (Reproduced from ref. 7 with permission.)... 

An ideal method for sequencing a peptide or protein would have a reagent that clips off just one amino acid at a time from the end of the chain, and identifies it. Such a method was devised by Pehr Edman (professor at the University of Lund in Sweden), and it is now widely used. 

The process can be repeated on the chain-shortened peptide to remove another amino acid residue. In this way, the amino acid residues are removed one at a time until the entire sequence has been identified. This entire process has been fully automated, and automated peptide sequencers are capable of sequencing a peptide chain with as many as 50 amino acid residues. 

Edman degradation identifies the N-terminal amino acid. Repeated Edman degradations can be used to sequence a peptide from the N-terminal end. 

Modern methods of amino-acid and peptide analysis, have enabled the complete amino-acid sequence of a number of proteins to be worked out. The grosser structure can be determined by X-ray diffraction procedures. Proteins have molecular weights ranging from about 6 000 000 to 5 000 (although the dividing line between a protein and a peptide is ill defined). Edible proteins can be produced from petroleum and nutrients under fermentation. 

Besides such textual databases that provide bibhographic information, sequence databases have attained an even more important role in biochemistry. Sequence databases are composed of amino add sequences of peptides or proteins as well as nudeotide sequences of nudeic acids. The 20 amino adds are mostly represented by a three-letter code or by one letter according to the biochemical conventions) the four nudeic adds are defined by a one-letter code. Thus the composition of a biochemical compound is searchable by text retrieval methods. 

There are several levels of pepfide sfrucfure The primary structure is the ammo acid sequence plus any disulfide links With the 20 ammo acids of Table 27 1 as building blocks 20 dipeptides 20 tripeptides 20" tetrapeptides and so on are possible Given a peptide of unknown structure how do we determine its ammo acid sequence" ... 

Only the N terminal amide bond is broken m the Edman degradation the rest of the peptide chain remains intact It can be isolated and subjected to a second Edman procedure to determine its new N terminus We can proceed along a peptide chain by beginning with the N terminus and determining each ammo acid m order The sequence is given directly by the structure of the PTH derivative formed m each successive degradation... 

A reevaluation of the original sequence data established that natural bradykinm was indeed the nonapeptide shown Here the synthesis of a peptide did more than confirm structure synthesis was instrumental m determining structure... 

The primary structure of a peptide is its ammo acid sequence We also speak of the secondary structure of a peptide that is the conformational relationship of nearest neighbor ammo acids with respect to each other On the basis of X ray crystallographic studies and careful examination of molecular models Linus Pauling and Robert B Corey of the California Institute of Technology showed that certain peptide conformations were more stable than others Two arrangements the a helix and the (5 sheet, stand out as... 

The primary structure of a peptide is given by its ammo acid sequence plus any disulfide bonds between two cysteine residues The primary structure is determined by a systematic approach m which the protein is cleaved to smaller fragments even individual ammo acids The smaller fragments are sequenced and the mam sequence deduced by finding regions of overlap among the smaller peptides... 

Synthesis of a peptide of prescribed sequence requires the use of pro tectmg groups to minimize the number of possible reactions... 

Primary carbon (Section 2 13) A carbon that is directly at tached to only one other carbon Primary structure (Section 27 8) The sequence of ammo acids in a peptide or protein... 

The sequence of amino acids in a peptide can be written using the three-letter code shown in Figure 45.3 or a one-letter code, both in common use. For example, the tripeptide, ala.ala.phe, could be abbreviated further to AAF Although peptides and proteins have chain-like structures, they seldom produce a simple linear system rather, the chains fold and wrap around each other to give complex shapes. The chemical nature of the various amino acid side groups dictates the way in which the chains fold to arrive at a thermodynamically most-favored state. 

However, interpretation of, or even obtaining, the mass spectrum of a peptide can be difficult, and many techniques have been introduced to overcome such difficulties. These techniques include modifying the side chains in the peptide and protecting the N- and C-terminals by special groups. Despite many advances made by these approaches, it is not always easy to read the sequence from the mass spectrum because some amide bond cleavages are less easy than others and give little information. To overcome this problem, tandem mass spectrometry has been applied to this dry approach to peptide sequencing with considerable success. Further, electrospray ionization has been used to determine the molecular masses of proteins and peptides with unprecedented accuracy. 

P-Endorphin. A peptide corresponding to the 31 C-terminal amino acids of P-LPH was first discovered in camel pituitary tissue (10). This substance is P-endorphin, which exerts a potent analgesic effect by binding to cell surface receptors in the central nervous system. The sequence of P-endorphin is well conserved across species for the first 25 N-terminal amino acids. Opiates derived from plant sources, eg, heroin, morphine, opium, etc, exert their actions by interacting with the P-endorphin receptor. On a molar basis, this peptide has approximately five times the potency of morphine. Both P-endorphin and ACTH ate cosecreted from the pituitary gland. Whereas the physiologic importance of P-endorphin release into the systemic circulation is not certain, this molecule clearly has been shown to be an important neurotransmitter within the central nervous system. Endorphin has been invaluable as a research tool, but has not been clinically useful due to the avadabihty of plant-derived opiates. 

Development of a peptide vaccine is derived from the identification of the immunodominant epitope of an antigen (141). A polypeptide based on the amino acid sequence of the epitope can then be synthesized. Preparation of a peptide vaccine has the advantage of allowing for large-scale production of a vaccine at relatively low cost. It also allows for selecting the appropriate T- or B-ceU epitopes to be included in the vaccine, which may be advantageous in some cases. Several vaccines based on peptide approaches, such as SPf66 (95) for malaria and an HIV-1 peptide (142) have been in clinical trials. No peptide vaccines are Hcensed as yet. 

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