Ovalbumin, tryptic digest

FIGURE 6.44 Two-dimensional separations of (a) an ovalbumin tryptic digest and (b) an ovalbumin tryptic digest spiked with a peptide of the sequence GGLEPINFQTAADQAR. The arrow indicates the identified peptide [565]. Reprinted with permission from the American Chemical Society. 

Figure 9.6 Surfer-generated chromatoeletropherogram of fluorescamine-labeled tryptic digest of ovalbumin. Reprinted from Analytical Chemistry, 62, M. M. Bushey and J. W. Jorgenson, Automated instrumentation for comprehensive two-dimensional high-performance liquid chromatography/capillary zone electrophoresis, pp 978-984, copyright 1990, with permission from the American Chemical Society.

FIGURE 1.3 Three-dimensional representation of a tryptic digest of ovalbumin. The three-dimensional separation consists of size-exclusion chromatography (first dimension), reversed-phase LC (second dimension), and capillary electrophoresis (third dimension). From Moore and Jorgenson, (1995) with permission of the American Chemical Society. 

FIGURE 8.7 2D chromatogram of a SEC x RPLC separation of a tryptic digest of ovalbumin. Reproduced with permission from Opiteck et al. (1997), copyright 1997, American Chemical Society. 

Two-dimensional SECxRP can be also used for protein and peptide separation, and this approach has been used for separation of peptides in tryptic digests of ovalbumin and serum albumin [14],... 

Fig. 4 The total ion chromatography (TIC) of the separation of a tryptic digest of chicken ovalbumin with a sample injection amount of 12 pmol corresponding to the original protein [52]. Column length, 6 cm. Conditions 20 min, 0-40% acetonitrile gradient 1000 V applied voltage with a 40-bar supplementary pressure. (From Ref. 52 reproduced with permission of the authors and the American Chemical Society.)...

The spectrophotometric evidence reviewed above for the binding of a proportion of the phenolic hydroxyl groups of the tyrosine residues of native proteins is supported by work on the action of tyrosinase on proteins. Sizer (1946) found that this enzyme oxidizes the tyrosine residues in native trypsin, pepsin, chymotrypsin, casein, peptone, insulin, and hemoglobin. Native ovalbumin, human and bovine serum albumin, tobacco mosaic virus (nucleoprotein), human y- and bovine /3-globulins, and bovine fibrinogen are not susceptible to tyrosinase, but become so after tryptic digestion. It was shown (Sizer, 1947) that for the proteins which are oxidized by tyrosinase in the native state, the observed reaction does indeed occur with the intact proteins and does not require preliminary degradation to tyrosine peptides or free tyrosine. The kinetics of the oxidation of tyrosine by tyrosinase have been studied spectropho-tometrically (Mason, 1948 etc.). 

Table 2. Peptides identified in the tryptic digest of native ovalbumin. Bold characters in the sequence column represent potential esterification sites. Last column (Ester) shows if the peptide was also found in the esterified sample listed in Table 4 ( + iffound, otherwise)...

Fig. 2. Electrochromatographic separation of peptides from a tryptic digest of ovalbumin on C,g-modified pillars (main picture), and part of a microfabricated pillar array showing the inlet region of the array (upper left). (Reprinted with permission from [11] and [26], copyright (1998) American Chemical Society and (1999) Elsevier).

The applicability of microfabricated pillar arrays (see Section 2.7.2.2.4) for the electrochromatographic separation of peptides from a tryptic digest of ovalbumin was demonstrated by He et al. [1 Ij. Their separation column was 4.5 cm long and... 

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