Peptides HPLC retention time

There are two separate but interrelated aspects to QSAR modeling of antibacterial peptides the choice of QSAR descriptors and the choice of numerical analysis techniques used to relate these values to antibacterial activity. A simple example of a QSAR descriptor is the total charge of a peptide. A large number of QSAR descriptors is available for small compounds in the literature and from commercial software products that may be considered. A smaller subset is used in QSAR studies of antibacterial peptides and may be separated into two categories descriptors based on empirical values and calculated descriptors. An example of an empirical value is HPLC retention time, which is a surrogate measure of solubility or hydrophilicity/hydrophobicity. An example of a calculated descriptor is total peptide charge at pH 7. 

We have gained an appreciation for the ionization bias observed between MALDI and LSIMS. The utilization of PSD to identify known peptides and provide sequence information has been investigated for conotoxins. This approach to obtaining sequence information on novel peptides is attractive because of the low amount of material required. A number of mass spectrometric based derivatizations have been used to scan fractions of venoms in order to characterize peptides of interest. For closely related components (based on HPLC retention time and mass), the small scale derivatization schemes can be used to test hypotheses about peptides with otherwise novel masses (i.e. which may be homologs). The mass accuracy of the TOF technique, with a gridless reflector, was important for identifying and calling these substitutions. 

Fifty-three peptide samples were submitted by 48 laboratories. Previous studies by this committee have demonstrated the need for multiple analytical methods for the assessment of purity. Therefore the peptides in this study were analyzed by AAA, HPLC, ESI-MS and MALDI-MS to determine purity (Table I). Only two peptide samples had less than 50% of the desired product, and three other samples had less than 70% of the desired product, as judged by their mass spectra, amino acid composition and HPLC retention time. Overall, the peptides were of excellent quality. 

Figure 7 Edman degradation sequence analysis, (a) Chromatograms for each sequence analysis cycle (b) [see p. 786] relative yield per cycle. The analysis was performed on a PE Biosystems cLC Precise 490 sequencer. Two microliters of an approximately 2 nM solution of the peptide in 0.2% TEA was applied to a PVDF membrane (0.4 X 0.4 mm) and allowed to air dry. Biobrene-T (2.5 /xL of a solution containing Biobrene-T/methanol/0.2% TEA, 1 7 2, v/v/v Biobrene-T from PE Biosystems) was then added on top of the dried peptide. Sequence anMysis of the dry sample on the membrane was accomplished by means of instrument protocols provided by the manufacturer. A standard mixture containing 19 PTH-amino acids (all of the common amino acids except Cys) was utilized to determine the HPLC retention times for each derivatized residue.

Peptide antibiotics include a wide range of compounds such as cycloserine (an amino acid), bacitracin (a cyclic peptide), thiostrepton (a Unear peptide), neocarzinostatin (a polypeptide) and bleomycin (a glycoprotein). These compounds possess either antibacterial and/or antitumor activity and should be regarded as conventional peptides for the purposes of HPLC. For a detailed overview of the HPLC retention times of the more common peptide antibiotics in various buffer systems the reader is referred to a recent review (Aszalos and Aquilar, 1984). The recommended stationary phase is a Vydac RP-18 column because it produced the least amount of peak taiUng. Mobile... 

AP(l-40) and N-cysteinyl-AP(l-40) peptides were synthesized by SPPS/Fmoc protection sttategy at a preparative scale (100 pM) and the products purified by HPLC (Figure 15.1). Ap(l-40) and Cys-AP(l-40) were separated at HPLC retention times of... 

Sasagawa, T. and Teller, D. C., Prediction of peptide retention times in re-versed-pahase HPLC, in CRC Handbook of HPLC for the Separation of Amino Acids, Peptides, and Proteins, Vol. II, Hancock, W. S., Ed., CRC Press, Boca Raton, FL, 1984, 53. 

A typical HPLC separation using a 15-cm column of 15,000 theoretical plates produces peak capacity (Giddings, 1991) of about 80-100 under isocratic conditions and up to 150 under gradient conditions in 1 h(Eq. 7.3, n peak capacity, A number of theoretical plates of a column, and fR and t retention time of the last and the first peak of the chromatogram, respectively). An increase in the number of separated peaks per unit time can be achieved by increased separation speed made possible by monolithic silica columns (Deng et al., 2002 Volmer et al., 2002). This has also been shown for peptides and proteins (Minakuchi et al., 1998 Leinweber et al., 2003). 

Cys(Acm)U5]-endothelin I [H-Cys(Acm)-Ser-Cys-Ser-Ser-Leu-Met-Asp-Lys-Glu-Cys-Val-Tyr-Phe-Cys(Acm)-His-Leu-Asp-Ile-Ile-Trp] (22 0.41 pmol) was treated with silver(I) trifluoroacetate/DMSO/ 1M HCI in an analogous manner to that described in Section 6.1.1.1, The HPLC elution pattern of the crude peptide showed two peaks, with retention times identical to those of endothelin-I and an isomer in a ratio of 8 1. Each product was purified by HPLC to yield endothelin-I (23) and its isomer (7.5% yield 62%) these products were characterized by HPLC, FAB-MS, and amino acid analyses. 

Note that proteins are now synthesized routinely by stepwise assembly or fragment condensation (see Vol. E22a, Section 4.1 ) 67 68 and chances of product contamination with shorter fragments that may have similar retention times under RP-HPLC should be considered 69 Additionally, size-exclusion HPLC was found to be particularly useful in distinguishing linear from cyclic peptides. 

Cobb and Novotny (7) obtained improved separations using C)8 microcolumns as a method for separating quantities on the order of 4 picomoles of tryptic peptides of phosphorylated and dephosphorylated /3-casein. Figure 4 shows two peaks with different retention times, corresponding to the phosphorylated and dephosphorylated forms of the same peptide. The rest of the peptide map is similar. Using this microcolumn, phosphorylation of a single amino acid on a protein can be detected. The method is reproducible with standard deviations smaller than 2%. Characterization of bovine /3-lg tryptic peptides by RP-HPLC on a Nucleosil Cl 8 column was also reported (123). 

Within a series of related peptides, retention time on RP-HPLC can be used as an indicator of the peptide s structure. For example, in a homologous series of GS14 diastereomers in which each peptide contains a different enantiomeric substitution within the GSM (2) framework, each peptide possesses a unique retention time on RP-HPLC as shown in Figure 3J12 ... 

The structure of peptides containing 20 eukaryotic natural amino acids is now routinely determined by the use of automatic protein microsequencer, which uses Edman chemistry to convert each a-amino acid sequentially to its phenylthiohydantoin (PTH) derivative. The formed PTH-amino acids can be identified by their retention times on HPLC systems by comparison with reference standards derived from the 20 natural amino acids. For an OBOC peptide library composed of natural amino acids, the sequencing protocols of the automatic sequencer are well developed and standardized. However, structure determination of peptides composed of unnatural a-amino acids requires modification of the standard sequencing program.32 For peptides composed of non-a-amino acids, one can use an encoding strategy or mass spectrometry if a cleavable linker is employed. In this chapter, we shall focus on the new sequencing method we have developed for unnatural a-amino acids. 

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