Release and analysis of polypeptides and glycopolypeptides from formalin-fixed, paraffin wax-embedded tissue.
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 glycopolypeptides 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.
Brooks
S A
SA
School of Biological and Molecular Sciences, Oxford Brookes University, Headington, Oxford, UK.
Dwek
M V
MV
Leathem
A J
AJ
D000818:Animals; D015153:Blotting, Western; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D003488:Cyanogen Bromide; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D005404:Fixatives; D005557:Formaldehyde; D006020:Glycopeptides / Q000032:analysis*; D007124:Immunoenzyme Techniques; D008099:Liver / Q000737:chemistry; D016612:Paraffin Embedding; D010455:Peptides / Q000032:analysis*; D051381:Rats; D017208:Rats, Wistar; D016707:Tissue Fixation; D014168:Transferrin / Q000032:analysis*
D005404:Fixatives; D006020:Glycopeptides; D010455:Peptides; D014168:Transferrin; D005557:Formaldehyde; D003488:Cyanogen Bromide