Peptides retrieval

Besides such textual databases that provide bibhographic information, sequence databases have attained an even more important role in biochemistry. Sequence databases are composed of amino add sequences of peptides or proteins as well as nudeotide sequences of nudeic acids. The 20 amino adds are mostly represented by a three-letter code or by one letter according to the biochemical conventions) the four nudeic adds are defined by a one-letter code. Thus the composition of a biochemical compound is searchable by text retrieval methods. 

Figure 7.6 Peptides do not denature after baking (dry heat) or deparaffinization and antigen retrieval (wet heat). Peptide-coupled slides were treated as indicated on the Y-axis and then immunohistochemically stained. In this particular example, an ER peptide with an ER MAb was used. The resulting peptide spot intensity (mean pixel intensity on a 1-256 scale) was measured and is shown on the y-axis.The data represent the means and SD or triplicate measurements. The experiments on the left (solid bars) and the right (hatched bars) were conducted at different times and have no connection to one another. Adapted with permission from Sompuram et al.6...

PEPTIDE CONTROLS CAN DETECT PROBLEMS WITH ANTIGEN RETRIEVAL... 

Figure 7.9 Appropriate antigen retrieval and immunostaining of peptide controls and tissue sections, stained for HER2. The tissue section on the left has an island of 3+ HER2 tumor, toward the top of the tissue section. The tissue section on the right does not express HER2. Identifying information on the label was removed. See color insert.

Figure 7.10 shows the result from a clinical laboratory that did not perform antigen retrieval correctly. The unfixed peptides at 50 and lOpg/mL stained correctly, but the fixed controls did not. The tissue section (left slide, labeled HER2-99 ) also did not stain well, since it requires antigen retrieval. 

Figure 7.10 Immunostain result with inadequate antigen retrieval, resulting in staining of unfixed but not fixed peptide controls. The HER2+ tumor (left slide) is largely unstained as well. See color insert.

Sompuram S, Vani K, Bogen S. A molecular model of antigen retrieval using a peptide array. Am. J. Clin. Pathol. 2006 125 91-98. 

Figure 16.1 Montage of images, after immunostaining of peptides. The antibody clones for these analytes are D07 (p53), 9C2 (HER2), 1D5 (ER), and 636 (PR). The peptides were spotted in duplicate, adjacent to each other. The left-hand column ( Not Fixed ) illustrates stained peptide spots that were not fixed, representing a baseline condition. The middle column was fixed in formalin and not antigen retrieved. The peptides for p53 and HER2 lost immunoreactivity whereas the peptides for ER and PR continued to be immunoreactive. The right-hand column of peptide spots were both formalin fixed and antigen retrieved. Adapted with permission from Reference 16, 2004 American Society for Clinical Pathology.

These initial findings do not exclude other possible formaldehyde-induced reactions with tissue proteins. Notably, this first model system was not designed to detect the role of lysine residues. Lysine has a propensity to react with and form a variety of different types of cross-links with other amino acids in the presence of formaldehyde.1,3 417 Therefore, it is likely to also be important in reactions with formaldehyde. In fact, peptides with internal lysine residues were purposefully excluded from this initial study for technical reasons. To explore the importance of lysine residues in antigen retrieval, an alternative method was employed. 

In order to model the effect of formalin fixation and antigen retrieval on antibody immunoreactivity, we used a peptide epitope array (Fig. 16.5). The peptide epitopes are derived from the exact sequences in the native proteins. In this experiment, the initial findings are similar to those shown in Figure 16.1, but they are then extended by allowing for the role of adjacent proteins. 

Figure 16.5 Immunostained peptide arrays after various treatments of fixation, protein cross-linking, and antigen retrieval, as indicated at the top. Each row has a different peptide that is immunoreactive for the antibody denoted to the left. Column A represents the baseline condition, without any treatment whatsoever. Column B shows immunoreactivity of each peptide after overnight formalin fixation. Column C shows the immunoreactivity after first coating the array with an irrelevant protein (casein) followed by overnight formalin fixation. Column D illustrates the immunoreactivity of the peptides after the treatment of column C, and then antigen retrieval. Reproduced with permission from Reference 15, 2006 American Society for Clinical Pathology.

A reasonable objection to any in vitro model is whether it accurately mirrors the actual process. A strength of this model is that the peptides in the array, mounted on the microscope glass slide, are the very same as the antibody epitopes in the native proteins. Therefore, the types of formaldehyde-induced chemical reactions at or near the epitope are the same as would likely occur in a tissue sample. An additional strength of the model is that the experimental data using the peptide array completely account for the loss of immunoreactivity after formalin fixation and the recovery of immunoreactivity after antigen retrieval (Fig. 16.5). Nonetheless, our data do not prove that the model accurately represents formaldehyde reactions in tissue specimens. For example, our data do not exclude other causes of steric interference. 

Even among the group of peptide epitopes, there was a striking variability in the speed of formalin fixation and antigen retrieval, suggesting hetero-... 

Figure 16.8 Intensity of immunohistochemical staining as a function of the length of antigen retrieval time. All values represent the mean of triplicate measurements. The staining intensity of a peptide array that was not formalin fixed is shown at the far right of the graph, as a control. Reproduced with permission from Reference 15, 2006 American Society for Clinical Pathology.

In the absence of further targeting information, the expressed protein is secreted to the apoplast. The stability of antibodies in the apoplast is lower than in the lumen of the ER. Therefore, antibody expression levels can be increased up to ten times higher if the protein is retrieved to the ER lumen using an H/KDEL C-terminal tetra-peptide tag [133]. Again, although the principles of ER-retention in molecular farming have been established using antibodies, it is likely that they will also apply to many other proteins. 

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