Individual Peptides

Peptides are widespread in nature. They are often involved in specific biological activities (peptide hormones, peptide toxins, peptide antibiotics). A number of peptides of interest to food chemists are outlined in the following sections. 

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An interesting approach has recently been chosen in the MBO(N)D program ([Moldyn 1997]). Structural elements of different size varying from individual peptide planes up to protein domains can be defined to be rigid. During an atomistic molecular dynamics simulation, all fast motion orthogonal to the lowest normal modes is removed. This allows use of ca. 20 times longer time steps than in standard simulations. 

A typical TIC chromatogram from an analysis of peptides resulting from enzymatic digest of myoglobin. The peaks represent individual peptides eluting from an LC column and being mass measured by a spectrometer coupled to it through a dynamic-FAB inlet/ion source. 

Figure 6.7 Formation of cross-linkage between individual peptide chains in the peptidoglycan layer of S. aureus.

Sequence information for the remaining fragments was obtained by Edman degradation (see Section 5.3.1 above) after isolation of the individual peptides using preparative HPLC - the chromatographic resolution being sufficient to allow this, and thus enabled the complete sequence to be determined. 

There have been notable successes in the replacement of individual peptide residues by peptoid monomers with retention of in vitro activity and enhancement of specificity. Unfortunately, attempts to completely transform those bioactive peptides that function via specific peptide-protein binding events into entirely pep-toid-based ohgomers have so far proven successful only at short chain lengths (e.g. [23]). It remains to be seen whether any general strategy can be developed in... 

Mixtures of three peptides were also screened for their ability to form fibrils containing all three. Rather than testing all possibilities, carefully selected mixtures were used. Peptides in the equimolar mixtures were charged, but the overall sum of the peptide charges was zero, to build in chain-chain electrostatic attractions. If individual peptides or a peptide pair in a mixture formed fibrils, the mixture was not studied. Application of these criteria led to six possible mixtures these were mixed at 4 pM each at pH 7.4 and left for several days at room temperature to self-assemble. 

Thioflavin binding indicated fibrils formed for two of these three mixtures (EEEK, EKEE, KKKK and EEKE, KEEE, KKKK). The requirement of all three peptides to produce fibrils was further demonstrated by testing individual peptides... 

Complex peptide mixmres can now be analyzed without prior purification by tandem mass spectrometry, which employs the equivalent of two mass spectrometers linked in series. The first spectrometer separates individual peptides based upon their differences in mass. By adjusting the field strength of the first magnet, a single peptide can be directed into the second mass spectrometer, where fragments are generated and their masses determined. As the sensitivity and versatility of mass spectrometry continue to increase, it is displacing Edman sequencers for the direct analysis of protein primary strucmre. 

Figure 2.5. Tandem mass spectrometry. A. A peptide mixture is electrosprayed into the mass spectrometer. Individual peptides from the mixture are isolated (circled peptide) and fragmented. B. The fragments from the peptide are mass analyzed to obtain sequence information. The fragments obtained are derived from the N or C terminus of the peptide and are designated "b" or "y" ions, respectively. The spectrum shown indicates peptides that differ in size by the amino acids shown.

Then the mass spectrum of the mentioned peptide mixture is measured. The set of molecular mass values (peak list) corresponding to individual peptides is characteristic for the protein and can be considered as its fingerprint. 

The sensitivity and the selectivity depend on the data quality. Figure 7.5c displays results from a simulation employing the same data set as was used in Fig. 7.5b, together with a simulation in which the MS-MS spectra have only five mass values corresponding to an individual peptide (out of 25). Hence we see that the sensitivity and the selectivity depend on the data quality. 

In 1961 Ansorge et al. (A5), using the same technique as previously, determined the peptide composition of urine derived from four normal subjects, three males and one female. Among twenty isolated peptides, seventeen were found in all specimens of urine, two peptides in three specimens, and the remaining one only in two specimens of urine. The identity of individual peptides was established on the basis of their electrophoretic and chromatographic behavior, as well as the amino acid composition after complete hydrolysis. It should be pointed out, however, that the amino acid composition of the peptides examined differs considerably from that obtained by the same authors in the case of the peptides described in 1958 (HI). 

The second method relies on the partial interpretation of the spectra. A short amino acid stretch of one to three amino acids is read out. This sequence, together with its precise mass location in the peptide of a given mass, is used to identify the whole peptide in the database (Mann, 1996). Since the algorithms depend only on individual peptides, expressed sequence tag databases can be searched successfully. 

Zorbas V, Ortiz-Acevedo A, Dalton AB, Yoshida MM, Dieckmann GR, Draper RK, Baughman RH, Jose-Yacaman M, Musselman IH (2004) Preparation and characterization of individual peptide-wrapped single-walled carbon nanotubes. J. Am. Chem. Soc. 126 7222-7227. 

Thymus-derived, or T, lymphocytes also have an immunoglobulin-like surface receptor linked with accessory proteins to form the T-cell receptor (TCR). There are similarities between the BCR and TCR in that the recognition part of the T receptor complex is also a dimer. In most T cells, the dimer consists of a and [3 individual peptide... 

In mammals, two groups of host defense peptides are prominent the cathelicidins (notably human LL-37, porcine protegrin, and bovine indolicidin) and the defensins (human a- and /3-defensins). These two classes of peptides are involved in both intracellular and extracellular defensive responses and as individual peptides can be synthesized and stored in either active or inactive form within cells or secreted through degranulation into the surrounding environment. ... 

The second approach is the tandem mass spectrometric method (Wilm et ah, 1996 Link et ah, 1999 Yates, 2000). This method relies on fragmentation of individual peptides in the tryptic peptide mixture to gain sequence information. Its main advantage is that sequence information derived from several peptides is much more specific for the identification of a protein than a list of peptide masses. The sequence data can be used to search not only protein sequence databases but also nucleotide databases such as expressed sequence tag (EST) databases and, more recently, even... 

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