Lysozyme surrogates

We found that solutions of hen egg white lysozyme, bovine ribonuclease A (RNase A), or a 1 2 mol ratio of bovine carbonic anhydrase lysozyme formed opaque gels within 2 min when mixed with an equal volume of 20% NBF.25,26 Multi-protein tissue surrogates comprised of 50% w/v lysozyme and up to four additional proteins have also been formed (Fowler et al., unpublished results). After overnight fixation, the surrogates were firm and sliced easily with a razor blade for sampling. To determine the optimal... 

TABLE 14.2 Effects of Detergent and pH on the Recovery of Protein from FFPE Lysozyme Tissue Surrogates... 

Notes Lysozyme tissue surrogate samples (1.5 mg) histologically processed to paraffin embedding were rehydrated and resuspended in the indicated recovery buffer. Total protein in the supernatants was assessed colorimetrically after heating at 100C for 20min, followed by 60C for 2h. The % recovery values are the mean, the standard deviation. For more detail, see Reference 25. 

Figure 14.3 SDS-PAGE of recovery of lysozyme in the presence of BME. Lane M, molecular weight marker lane 1, FFPE lysozyme tissue surrogate lane 2, a 75 mg/mL solution of lysozyme heat coagulated for 10 min at 100°C in 10 mM sodium phosphate buffer, pH 7.4. Both preparations were resuspended in 20mM Tris-HCl, pH 4, with 2% SDS and 0.5 M BME, and heated at 100°C for 20min followed by a cycle of heating at 60°C for 2h. For more detail, see Reference 25.

Figure 14.4 Gel image of proteins extracted from a mixed carbonic anhydrase lysozyme tissue surrogate. Lane M, molecular weight marker lane 1, a 1 2 mol ratio mixture of native, non-formalin-treated carbonic anhydrase and lysozyme lane 2, mixed surrogate with 1 2 mol ratio carbonic anhydrase lysozyme, solubilized and retrieved in 20mM Tris-HCl, pH 4.0, with 2% SDS lane 3, mixed surrogate with 1 2 mol ratio carbonic anhydrase lysozyme, solubilized and retrieved in 20mM Tris-HCl, pH 6.0, with 2% SDS. Protein bands corresponding to lysozyme monomer (a), carbonic anhydrase monomer (b), and the putative lysozyme-carbonic anhydrase heterodimer (c) are indicated. For more detail, see Reference 25.

The tissue surrogates described here clearly represent a simplification of real FFPE tissues. However, they represent a useful and efficient construct for the evaluation and optimization of tissue extraction conditions for proteomic studies. More informative studies will likely be realized by using more complex tissue surrogates, which can be created by incorporating additional proteins into lysozyme solutions. Tissue surrogates comprised of up to five proteins have been successfully analyzed by MS (Fowler, unpublished data). Additionally, RNA, DNA, lipids, or carbohydrates can be added at nanomolar to millimolar concentrations to increase the complexity of the model system to better mimic whole tissue. The use of these more complex tissue surrogates should facilitate the development of protein recovery protocols optimal for proteomic investigation. 

TABLE 19.2 Recovery of Protein from a Lysozyme Tissue Surrogate... 

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