Error peptides

The purity of the immunogen is of controversial importance. For synthetic substances, however, no argument exists—the likelihood of closely related substances (such as error peptides ) being present in im-... 

F-nmr spectroscopy of trifluoracetylated solid-phase peptide products has been found to be a very sensitive method for the detection of error peptides (Bayer et al., 1972). The F-chemical shift of acylated peptides is very sensitive to the nature of the modified residue. 

In the case of smaller peptides, highly pure products have been obtained in good yields by simple evaporation of filtrate from the resin-peptide cleavage product. If necessary, the product peptide can be purified by ion-exchange chromatography to separate the error-peptides ef-... 

Formation of error-peptides Weygand and Obermeier (1968) and Bayer et aL (1970a) have divided the error-peptides (peptides other than those desired) into two classes (a) truncated sequences and (b) failure sequences. When a peptide chain on the resin fails to grow beyond a certain length, it is termed a truncated sequence. In a failure sequence, one or more amino acids may be missing from the desired peptide sequences. 

Whereas in the solution method, the intermediate peptides are purified after each coupling reaction, thus eliminating the unreacted material and by-products, in the solid-phase method, the error-peptides keep accumulating on the resin making their separation after cleavage very difficult. 

Formation of error-peptides due to interchain aminolysis It has been established (Crowley and Rapoport, 1976) that low (<2%) cross-linked resins are not very rigid in the solvated state. The interchain aminolysis of N-blocked peptides has been observed (Beyerman et aL, 1972) and results in the formation of error-peptides by chain dimerization. In certain spe-... 

P2j Z = 2 DX = 1.43 R = 0.067 for 1269 intensities. The uracil residue is in the anti (63.4°) disposition. The conformation of the D-ribosyl group is 2T3 (176.8°, 37.5°). The orientation about the exocyclic, C-4 -C-5 bond is t (—174.2°). The phenyl and uracil ringsofthe same molecule lie in almost parallel planes, 120 pm apart. The phenyl group is disordered. The uracil ring is sandwiched by the phenyl rings, and vice versa. The 0-1 and N-a atoms of the peptide backbone are hydrogen-bonded to 0-4 and N-3 of atranslationally related uracil to form cyclic dimers. Such interactions serve as models for nucleic acid-protein interactions. [Coordinate errors H(02 ) x should be —1574, instead of —1474 H(Na)2 z should be —145 instead of— 645.]... 

To summarize, we have systematically tested all possible three- and two-layer ONIOM combinations of high-level QM (HQ=B3LYP/6-31G ), low-level QM (LQ=AM1), and MM (Amber) for the deprotonation energy and structure of a test molecule, an ionic form of a peptide. We find the errors introduced in the ONIOM approximation, in comparison with the target HQ (or HQ HQ HQ) calculation, generally increases in the order ... 

Mann, M. Wilm, M. Error-tolerant identification of peptides in sequence databases by peptide sequence tags. Anal. Chem. 1994, 66,4390-4399. 

Fig. 9. The 3/Hn coupling constants versus temperature for different alanine residues in XAO peptide. The reversibility of 3/hn coupling constant versus temperature was checked by measurements with temperature increasing from 2° to 56°C (labeled as heating) or decreasing from 56° to 6°C (labeled as cooling). The errors are the same in heating and cooling measurements the error bars are shown only for heating measurements for clarity. The conditions were temperature from 2° to 56°C, concentration ca. 4 mM, in 30 mM sodium acetate buffer (pH = 4.6, 10% D20 ). From Shi et al. (2002). Proc. Natl. Acad. Sd. USA 99, 9190-9195, 2002 National Academy of Sciences, USA.

Figure 3.4 Improvement of the activity of chimeric NRPSs using directed evolution. (1) A heterologous A domain is swapped into an NRPS, typically resulting in a significant loss of synthetase activity. (2) A library of chimeric synthetase mutants is constructed in which the heterologous A domain has been diversified (for example, by error-prone PCR). (3) The library is subjected to an in vivo screen for production of the unnatural nonribosomal peptide derivative. (4) Clones showing improved production are characterized and subjected to further rounds of diversification and screening...

Fig. n.i. Human duodenal expression variability of peptide and amino acid transporters (our unpublished data). Shaded box indicates 25-75% of expression range, the line within the box marks the median, and error bars indicate 10-90% of expression range. PEPT1, di-, tri-peptide transporter HPT1 (Ll-cadherin) peptide transporter SLC3A1, cystine, dibasic and neutral amino acid transporter y+LATl, cationic amino acid transporter ATBq, neutral... 

Several algorithms are available for the analysis of MS/MS spectra including SEQUEST, MASCOT, and X Tandem among others. Note that additional secondary quality control of assessment of MS/MS data has recently been implemented to assess identification probabilities and false positivity rates. The MS/MS spectra from an experiment can be interrogated against a concatenated forward and reverse database and an assessment of the intrinsic error rate of the data set can be made. Other approaches for secondary analysis of matching scores for peptide sequencing data include XCorr score normalization routines that are independent of peptide and database size.33... 

M. Mann and M. Wilm. Error-Tolerant Identification of Peptides in Sequence Databases by Peptide Sequence Tags. Anal. Chem., 66(1994) 4390-4399. 

Continuous ion series are often generated when multiply charged peptide ions are fragmented. The problem in de novo sequencing with electrospray tandem mass spectrometry lies in minimizing the error rate of the interpretation. There are two different approaches to this problem ... 

The extent of formation of protein disulfides with time was determined by withdrawing aliquots which were acidified to pH 5.5 and alkylated with N-ethylmaleimide. The disulfide content of the peptide was determined after its isolation. Formation of two intrapeptide disulfide bonds proceeded at the same rate (within experimental error) as formation of the first two disulfides in reduced lysozyme. The first-order rate constant for these two processes (0.5 min-1) was eight times that describing the rate of oxidation of reduced lysozyme in the presence of 6 M guanidinium chloride, suggesting substantial specificity in the process in absence of denaturant. An additional indication of specificity was the finding that 13-105 reached its maximum of two —S—S— bonds in less than 20 minutes, retaining one reduced thiol from 20 to 240 minutes. For subsequent studies this material was S-alkylated with N-ethylmaleimide. 

---