Analysing proteins from tissue samples that have been formalin-fixed and paraffin-embedded (FFPE) can provide critical information about how cells function before fixation. “When you formalin-fix the protein, you fix it in time; it is not going anywhere,” says Peter Tunon, vice-president of sales and marketing at Expression Pathology in Gaithersburg, Maryland. Researchers are taking advantage of this fact to explore the protein world in FFPE samples.

Expression Pathology was founded in 2001 by researchers from the US National Cancer Institute and the company Life Technologies (now Invitrogen) who had experience in studying gene expression in tissue and histology. “The company was founded on the fact that examining protein expression is crucial to understanding what is happening in cells,” says Tunon. To this end, Expression Pathology has worked on ways of extracting and isolating proteins from FFPE tissue samples for analysis by mass spectrometry or reverse-phase dot blot arrays. The company developed a technology that integrates extraction of total proteins from FFPE samples with tryptic digestion so that finished samples are ready for mass spectrometry. To simplify the system further, all reagents are completely compatible with mass-spectrometry instrumentation, says Tunon, making it a good starting point for broad-based screening of proteins in FFPE samples. Other companies also think that protein isolation from FFPE tissue samples will provide valuable information. QIAGEN and EMD Chemicals, both based in San Diego, California, now offer systems that chemically reverse formalin crosslinking to isolate full-length proteins for applications such as western blotting and protein arrays.

Caught on camera: a section of human muscle after dual histochemistry. Credit: LEICA MICROSYSTEMS

Surprisingly, post-translational modifications (PTMs) such as phosphorylation and acetylation, can also be observed when examining proteins isolated from FFPE samples. PTMs can be critical to the role of a protein in the cell, changing the function or localization. “We do see post-translational modifications that are preserved on the peptides, and they seem to be present in ratios that are similar to those we get when using fresh frozen tissues,” Tunon says of work done by Expression Pathology.

Even much older archival samples of FFPE tissues do not seem to pose a problem for protein extraction. “We have worked with samples that are more than 15 years old and found no difference in the profiles compared to samples that were just a few weeks old,” says Tunon. He is quick to add that he believes even older samples could be examined, but Expression Pathology has yet to test this idea.

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