Peptides from Columns

A small conductivity cell attached to the bottom of a column may also be used to locate the bands of amino acids and peptides, which will cause changes in the conductivity of the solution (Randall and Martin, 1949). 

Recently Drake (1950) has described a polarographic method for following the chromatography of proteins. Only substances containing cystine or cysteine will be detected but it may be useful where large peptides are being studied. The method is unaffected by changes in the solvent or salt concentration. 

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Cyclic peptide from 11 amino acids. Preparation by fermentation of Tolypocladium inflatum Gams with addition of DL-a-aminobutyric acid to the fermentation medium. Isolation by homogenization of mycelium, extraction with 90 % methanol and column chromatographic purification. 

Once the resolution has been optimized as a function of gradient rate, one can continue to fine-tune the separation, raising flow rate and temperature. In a study of temperature and flowrate variation on the separation of the tryptic peptides from rabbit cytochrome c, column performance doubled while analysis time was reduced by almost half using this strategy.97 Commercially available software has been developed to aid in optimization. As a final note, in an industrial laboratory optimization is not completed until a separation has been shown to be rugged. It is a common experience to optimize a separation on one column, only to find that separation fails on a second column of identical type. Reproducibility and rigorous quality control in column manufacture remains a goal to be attained. 

FIGURE 16.2 Representative base peak electropherograms from CZE runs of RPLC fractions, (a) Fraction 15 (5 peptide identifications) and (b) fraction 20 (19 peptide identifications). Column, bare fused silica capillary, 60 cm x 180 pm ODx30pm i.d. separation voltage, 15 kV observed CZE current, 1.91 p.A running electrolyte, 200 mm acetic acid + 10% isopropanol temperature, 22°C injection time, 10 s at 2 psi ( 4 nL total injection volume) supplementary pressure, 2 psi flow rate, 25nL/min spray voltage, 1.5 kV (reprinted with permission from Electrophoresis). 

Monitor the elution of reduced peptide from the column by measuring the absorbance at 280 nm (if peptide absorbs at this wavelength) as well as by performing an Ellman s assay (Section 4.1, this chapter) for sulfhydryl groups using a small aliquot (10-20 pi) of each collected fraction. 

Figure 16.4 A more advanced ICAT design uses an acid-cleavable spacer arm to facilitate elution of labeled peptides from a (strept)avidin affinity column. The use of 14C isotopes instead of deuterium labels permits precise reverse phase separations prior to mass spec that show no elution peak time differences between isotope-labeled and normal atom-labeled peptides.

Figure 4.13 Separation of the tryptic peptides from recombinant HGH using a 120-min linear gradient at 60°C from water + 0.1% TFA to 40 60 water acetonitrile + 0.1% TFA. Column Zorbax SB-C8, 4.6 X 150 mm, 30-nm pore, 5- tm particle size. (Reprinted from W.S. Hancock, R.C. Chloupek, J.J. Kirkland, and L.R. Snyder, J. Chromatogr. A, 686 31 [1994]. With permission from Elsevier Science.)...

Figure 2 HPLC Profile of Crude Base Peptide (a) H-Cys(Acm)84-Val124 Cys(Acm)95110-OH from the Addition of Segments 1 and 2. Peak (b) was Identified as the Base Peptide+ Hmb. Column Vydac C8 (250 x 4.6 mm) Eluant 15-40% Solution B in Solution A Linear Gradient for 25 min, Solvents as Figure 1 Detection 215 nm...

The need for enhanced detection sensitivity and automation has steadily increased for the separation and analysis of peptides from natural sources or proteolytic digestion of low abundance proteins this is also partly a consequence of the greater usage of combinatorial solid-phase synthetic approaches. Narrow bore (1-2 mm i.d.), microbore (0.5-1.0 mm i.d.), and capillary (100-500 pm i.d.) columns have provided attractive solutions to these problems. 1221 An important attribute of very small particle diameter micropellicular, porous, or nonporous RPC sorbents is that they are ideally suited to such microbore or capillary... 

Table 3 (73) compares the retention coefficients for synthetic peptides from various sources. To ensure comparability, the data has been standardized with respect to lysine and assigned a value of 100. The table shows that there are discrepancies between the results obtained using different chromatographic systems. Predictions of retention times should therefore be made using chromatographic systems similar to those used to calculate the retention coefficients for the amino acids. Casal et al. (75a) have made a comparative study of the prediction of the retention behavior of small peptides in several columns by using partial least squares and multiple linear regression analysis. 

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