Abstract
Archival tissue specimens are commonly stored as formalin- fixed, paraffin wax-embedded blocks. Formalin fixation facilitates excellent morphological preservation, and the immunoreactivity of many antigens is preserved, but formalin- induced chemical cross-linking of proteins renders them insoluble and inaccessible to standard biochemical extraction and analytical methods. Thus, biochemical analysis of tissue components identified by histochemistry, with the advantage of long-term clinical follow-up, is precluded. We have applied cyanogen bromide cleavage, a technique used routinely for fragmenting proteins for sequencing experiments, to solubilize transferrin polypeptides and glyco-polypeptides from formalin- fixed, paraffin wax-embedded rat liver. Cyanogen bromide cleaves protein specifically at methionine residues, yielding a predictable array of polypeptide fragments. Subsequent oligosaccharide analysis of the transferrin glycopolypeptides by anion exchange chromatography confirmed that, in addition to successful release of polypeptide chains, sialylated oligosaccharide structures remained intact after cyanogen bromide cleavage. This approach may have wide applicability to a range of research interests in which correlation of tissue biochemistry with long-term follow-up is advantageous.
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Anderson, B.F., Baker, H.M., Norris, G.E., Rice, D.W. & Baker, E.N. (1989) Structure of human lactoferrin: crystallographic structure analysis and refinement at 2.8 A resolution. J. Mol. Biol. 209, 711-34.
Crichton, R.R & Charloteaux-Wauters, M. (1987) Iron transport and storage. Eur. J. Biochem. 164, 485-506.
Clark, R.K. & Damjanov, I. (1986) Immunoblotting of keratin polypeptides extracted from tissues preserved in standard histologic fixatives. J. Histochem. Cytochem. 34, 679-82.
Dwek, M.V., Brooks, S.A., Streets, A. J., Harvey, D. J. & Leathem, A.J.C. (1996) Oligosaccharide release from frozen and paraffin-wax-embedded archival tissues. Anal. Biochem. 242, 8-14.
Gordon, A.H. & Louis, L.N. (1963) Preparation and properties of rat transferrin. Biochem. J. 88, 409-14.
Hara, A., Sakai, N., Yamada, H., Yoshima, N., Tanaka, T. & Mori, H. (1993) Immunoblot analysis of the placental form of glutathione-S-transferase in protein extracted from paraffin-embedded human glioma tissue. J. Cancer Res. Clin. Oncol. 119, 493-6.
Kobata, A. & Endo, T. (1993) Release of N-linked oligosaccharides and fractionation by gel filtration. In: Glycobiology - a Practical Approach (edited by Fukuda, M. & Kobata, A.), pp. 86-8. New York: IRL Press.
Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-5.
Lalani, E.N., Golding, M., Hudson, M., Chieffi, G., Stamp, G., Anilkumar, T.V., Sarraf, C. & Alison, M. (1995) Protein extraction and Western blotting from methacarn-fixed tissue. J. Pathol. 177, 323-8.
Layfield, R., Bailey, K., Lowe, J., Allibone, R., Mayer, R.J. & Landon, M. (1996) Extraction and protein sequencing of immunoglobulin light chain from formalin-fixed cerebrovascular amyloid deposits. J. Pathol. 180, 455-99.
Matsudaira, P. (1990) Limited N-terminal sequence analysis. Methods Enzymol. 182, 602-13.
Morgan, E.H. (1969) Factors affecting the synthesis of transferrin byrat tissue slices. J. Biol.Chem. 244, 4193-265.
Schagger, H. & Von Jagow, G. (1987) Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal. Biochem. 166, 368-79.
Schrieber, G., Dryburgh, H., Millership, A., Matsuda, Y., Inglis, A., Phillips, J., Edwards, K. & Maggs, J. (1979) The synthesis and secretion of rat transferrin. J. Biol. Chem. 254, 12013-19.
Tomita, M., Furthmayr, H. & Marchesi, T. (1978) Primary structure of human erythrocyte glycophorin A. Isolation and characterization of peptides and complete amino acid sequence. Biochemistry 17, 4756-70.
Walker, J.F. (1964) Formaldehyde. 3rd. edn. New York: Chapman & Hall.
Wessel, D. & FlÜgge, U. I. (1984) A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids. Ann. Biochem. 138, 141-3.
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Brooks, S.A., Dwek, M.V. & Leathem, A.J. Release and Analysis of Polypeptides and Glycopolypeptides from Formalin-fixed, Paraffin Wax-embedded Tissue. Histochem J 30, 609–615 (1998). https://doi.org/10.1023/A:1003222931605
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DOI: https://doi.org/10.1023/A:1003222931605