Jeffrey T. Mason

A liposome-PCR assay for the ultrasensitive detection of biological toxins

We describe an ultrasensitive immunoassay for detecting biotoxins that uses liposomes with encapsulated DNA reporters, and ganglioside receptors embedded in the bilayer, as a detection reagent. After immobilization of the target biotoxin by a capture antibody and co-binding of the detection reagent, the liposomes are ruptured to release the reporters, which are quantified by real-time PCR. Assays for cholera and botulinum toxins are several orders of magnitude more sensitive than current detection methods.

Modeling formalin fixation and antigen retrieval with bovine pancreatic ribonuclease A: I-structural and functional alterations.

Understanding the chemistry of protein modification by formaldehyde is central to developing improved methods to recover proteins from formalin-fixed paraffin-embedded tissues for proteomic analysis and to improve protein immunoreactivity for immunohistochemical studies. We used biophysical techniques to investigate the effects of formaldehyde treatment on bovine pancreatic ribonuclease A (RNase A). Treatment of RNase A with formaldehyde was shown by gel electrophoresis to lead to the rapid formation of intra- and intermolecular protein cross-links. Thermal studies revealed that these protein cross-links significantly increased the thermal denaturation temperature of RNase A preparations. Analysis of formaldehyde-treated RNase A oligomers isolated by gel chromatography revealed that intramolecular protein cross-links are primarily responsible for the increase in protein thermostability. Formaldehyde treatment also lowered the isoelectric point of the enzyme from 9.45 to the 6.0–7.4 range. Optical spectroscopic studies demonstrated that the formaldehyde-induced modifications did not significantly alter the secondary or tertiary structure of RNase A. Heating formaldehyde-treated RNase A at 65°C resulted in a significant reversal of the protein intra- and intermolecular cross-links and led to a partial restoration of enzymatic activity.

'Tissue surrogates' as a model for archival formalin-fixed paraffin-embedded tissues.

High-throughput proteomic studies of archival formalin-fixed paraffin-embedded (FFPE) tissues have the potential to be a powerful tool for examining the clinical course of disease. However, advances in FFPE tissue-based proteomics have been hampered by inefficient methods to extract proteins from archival tissue and by an incomplete knowledge of formaldehyde-induced modifications in proteins. To help address these problems, we have developed a procedure for the formation of ‘tissue surrogates’ to model FFPE tissues. Cytoplasmic proteins, such as lysozyme or ribonuclease A, at concentrations approaching the protein content in whole cells, are fixed with 10% formalin to form gelatin-like plugs. These plugs have sufficient physical integrity to be processed through graded alcohols, xylene, and embedded in paraffin according to standard histological procedures. In this study, we used tissue surrogates formed from one or two proteins to evaluate extraction protocols for their ability to quantitatively extract proteins from the surrogates. Optimal protein extraction was obtained using a combination of heat, a detergent, and a protein denaturant. The addition of a reducing agent did not improve protein recovery; however, recovery varied significantly with pH. Protein extraction of >80% was observed for pH 4 buffers containing 2% (w/v) sodium dodecyl sulfate (SDS) when heated at 100°C for 20 min, followed by incubation at 60°C for 2 h. SDS-polyacrylamide gel electrophoresis of the extracted proteins revealed that the surrogate extracts contained a mixture of monomeric and multimeric proteins, regardless of the extraction protocol employed. Additionally, protein extracts from surrogates containing carbonic anhydrase:lysozyme (1:2 mol/mol) had disproportionate percentages of lysozyme, indicating that selective protein extraction in complex multiprotein systems may be a concern in proteomic studies of FFPE tissues.

Modeling formalin fixation and antigen retrieval with bovine pancreatic RNase A II. Interrelationship of cross-linking, immunoreactivity, and heat treatment

In this study, gel electrophoresis and capture enzyme-linked immunosorbent assay were used to assess the effect of formaldehyde treatment on the structural and immunological properties of bovine pancreatic ribonuclease A (RNase A). Prolonged incubation of RNase A in a 10% formalin solution leads to the formation of extensive intra- and intermolecular cross-links. However, these formaldehyde cross-links do not completely eliminate the recognition of RNase A by a polyclonal antibody. Comparative immunotitration of monomers, dimers, and oligomers greater than pentamers isolated from formalin-treated RNase A demonstrated that reduction of immunoreactivity due to intramolecular modifications prevails over the excluded volume effect of intermolecular cross-links. The latter only becomes important for intermolecular cross-links involving four or more molecules. The restoration of RNase A immunoreactivity during heating correlates with the reversal of formaldehyde cross-links if the incubation temperature does not exceed the denaturation temperature of the formalin-treated RNase A preparation. We conclude that formaldehyde cross-links stabilize antigens against the denaturing effects of high temperature, but the reversal of these cross-links is necessary for the restoration of immunoreactivity.

Modeling formalin fixation and histological processing with ribonuclease A: effects of ethanol dehydration on reversal of formaldehyde cross-links

Understanding the chemistry of protein modification by formaldehyde fixation and subsequent tissue processing is central to developing improved methods for antigen retrieval in immunohistochemistry and for recovering proteins from formalin-fixed, paraffin-embedded (FFPE) tissues for proteomic analysis. Our initial studies of single proteins, such as bovine pancreatic ribonuclease A (RNase A), in 10% buffered formalin solution revealed that upon removal of excess formaldehyde, monomeric RNase A exhibiting normal immunoreactivity could be recovered by heating at 60°C for 30 min at pH 4. We next studied tissue surrogates, which are gelatin-like plugs of fixed proteins that have sufficient physical integrity to be processed using normal tissue histology. Following histological processing, proteins could be extracted from the tissue surrogates by combining heat, detergent, and a protein denaturant. However, gel electrophoresis revealed that the surrogate extracts contained a mixture of monomeric and multimeric proteins. This suggested that during the subsequent steps of tissue processing protein–formaldehyde adducts undergo further modifications that are not observed in aqueous proteins. As a first step toward understanding these additional modifications we have performed a comparative evaluation of RNase A following fixation in buffered formaldehyde alone and after subsequent dehydration in 100% ethanol by combining gel electrophoresis, chemical modification, and circular dichroism spectroscopic studies. Our results reveal that ethanol-induced rearrangement of the conformation of fixed RNase A leads to protein aggregation through the formation of large geometrically compatible hydrophobic β-sheets that are likely stabilized by formaldehyde cross-links, hydrogen bonds, and van der Waals interactions. It requires substantial energy to reverse the formaldehyde cross-links within these sheets and regenerate protein monomers free of formaldehyde modifications. Accordingly, the ethanol-dehydration step in tissue histology may be important in confounding the successful recovery of proteins from FFPE tissues for immunohistochemical and proteomic analysis.

The Effect of Formaldehyde Fixation on RNA: Optimization of Formaldehyde Adduct Removal

Formalin-fixed, paraffin-embedded tissues generally provide low yields of extractable RNA that exhibit both covalent modification of nucleic acid bases and strand cleavage. This frustrates efforts to perform retrospective analyses of gene expression using archival tissue specimens. A variety of conditions have been reported to demodify formaldehyde-fixed RNA in different model systems. We studied the reversal of formaldehyde fixation of RNA using a 50 base RNA oligonucleotide and total cellular RNA. Formaldehyde-adducted, native, and hydrolyzed RNA species were identified by their bioanalyzer electrophoretic migration patterns and RT-quantitative PCR. Demodification conditions included temperature, time, buffer, and pH. The reversal of formaldehyde-fixed RNA to native species without apparent RNA hydrolysis was most successfully performed in dilute Tris, phosphate, or similar buffers (pH 8) at 70°C for 30 minutes. Amines were not required for efficient formaldehyde demodification. Formaldehyde-fixed RNA was more labile than native RNA to treatment with heat and buffer, suggesting that antigen retrieval methods for proteins may impede RNA hybridization or RNA extraction. Taken together, the data indicate that reliable conditions may be used to remove formaldehyde adducts from RNA to improve the quality of RNA available for molecular studies.

Conversions of Formaldehyde-modified 2′-Deoxyadenosine 5′-Monophosphate in Conditions Modeling Formalin-fixed Tissue Dehydration

Formalin-fixed, paraffin-embedded specimens typically provide molecular biologists with low yields of extractable nucleic acids that exhibit extensive strand cleavage and covalent modification of nucleic acid bases. This study supports the idea that these deleterious effects are promoted by the first step in formalin-fixed tissue processing—i.e., tissue dehydration with a graded series of alcohols. We analyzed the conversions of formaldehyde-modified 2′-deoxyadenosine 5′-monophosphate (dAMP) by reverse-phase ion-pair, high-performance liquid chromatography and found that dehydration does not stabilize N-methylol groups in the modified nucleotide. Furthermore, spontaneous demodification in a dry state or in anhydrous ethanol can be as fast as it is in aqueous solutions if the preparation is contaminated with salts of orthophosphoric acid. In ethanol, orthophosphates also catalyze formation of abundant N6-ethoxymethyl-dAMP, as well as cross-linking and depurination of nucleotides present in the mixture. Identification of the products was performed using ultraviolet absorbance spectroscopy and electrospray ionization Fourier-transform ion cyclotron resonance mass spectrometry. Alternatives to the traditional processing of formalin-fixed tissues are discussed.

Elevated hydrostatic pressure promotes protein recovery from formalin-fixed, paraffin-embedded tissue surrogates.

High-throughput proteomic studies on formalin-fixed, paraffin-embedded (FFPE) tissues have been hampered by inefficient methods to extract proteins from archival tissue and by an incomplete knowledge of formaldehyde-induced modifications to proteins. We previously reported a method for the formation of ‘tissue surrogates’ as a model to study formalin fixation, histochemical processing, and protein retrieval from FFPE tissues. In this study, we demonstrate the use of high hydrostatic pressure as a method for efficient protein recovery from FFPE tissue surrogates. Reversal of formaldehyde-induced protein adducts and crosslinks was observed when lysozyme tissue surrogates were extracted at 45 000 psi and 80–100°C in Tris buffers containing 2% sodium dodecyl sulfate and 0.2 M glycine at pH 4. These conditions also produced peptides resulting from acid-catalyzed aspartic acid cleavage. Additives such as trimethylamine N-oxide or copper (II) chloride decreased the total percentage of these aspartic acid cleavage products, while maintaining efficient reversal of intermolecular crosslinks in the FFPE tissue surrogates. Mass spectrometry analysis of the recovered lysozyme yielded 70% sequence coverage, correctly identified all formaldehyde-reactive amino acids, and demonstrated hydrolysis at all of the expected trypsin cleavage sites. This study demonstrates that elevated hydrostatic pressure treatment is a promising approach for improving the recovery of proteins from FFPE tissues for proteomic analysis.

Protein fixation and antigen retrieval: chemical studies

Abstract Fixation with formaldehyde is the first process to which most biopsy and necropsy specimens are exposed prior to dehydration and embedding in paraffin wax. Tissue specimens that have been fixed in formaldehyde have architectural characteristics that are familiar to virtually every pathologist and these facilitate routine diagnosis. Nevertheless, formaldehyde fixation has some deleterious effects including reduction in immunoreactivity and degradation of nucleic acids. Development of methods to counteract these deleterious effects requires an understanding of the chemical events that occur during tissue fixation and subsequent tissue processing. This short review illustrates some of the chemical consequences of formaldehyde fixation and ethanol dehydration. It also provides some insight into the molecular events accompanying heat-induced antigen retrieval.

Magnetic resonance microscopy of collagen mineralization.

A model mineralizing system was subjected to magnetic resonance microscopy to investigate how water proton transverse (T(2)) relaxation times and magnetization transfer ratios can be applied to monitor collagen mineralization. In our model system, a collagen sponge was mineralized with polymer-stabilized amorphous calcium carbonate. The lower hydration and water proton T(2) values of collagen sponges during the initial mineralization phase were attributed to the replacement of the water within the collagen fibrils by amorphous calcium carbonate. The significant reduction in T(2) values by day 6 (p < 0.001) was attributed to the appearance of mineral crystallites, which were also detected by x-ray diffraction and scanning electron microscopy. In the second phase, between days 6 and 13, magnetic resonance microscopy properties appear to plateau as amorphous calcium carbonate droplets began to coalesce within the intrafibrillar space of collagen. In the third phase, after day 15, the amorphous mineral phase crystallized, resulting in a reduction in the absolute intensity of the collagen diffraction pattern. We speculate that magnetization transfer ratio values for collagen sponges, with similar collagen contents, increased from 0.25 +/- 0.02 for control strips to a maximum value of 0.31 +/- 0.04 at day 15 (p = 0.03) because mineral crystals greatly reduce the mobility of the collagen fibrils.