Toward Improving the Proteomic Analysis of Formalin-fixed, Paraffin-embedded Tissue

Carol B Fowler; Timothy J O'Leary; Jeffrey T Mason


Expert Rev Proteomics. 2013;10(4):389-400. 

In This Article

Formaldehyde as a Fixative

Formalin fixation remains the preeminent technique for processing biopsy specimens for pathologic examination, the study of tissue morphology and archival preservation. Formaldehyde was first identified by Ferdinand Blum as a fixative for tissues more than a century ago.[13] For tissue processing, 37% (w/v) aqueous formaldehyde is diluted 1:10 to a yield a 3.7% (w/v) formaldehyde solution known as formalin. Formalin is often referred to as 10% formaldehyde in histology laboratories based upon the 1:10 volume dilution. Formalin used for tissue processing often contains approximately 1% (v/v) methanol as a stabilizer to prevent polymerization. A buffer, usually phosphate buffer (pH 7–7.4), is also typically included to suppress the formation of formic acid, which can result in the formation of acid formaldehyde-hematin precipitates. Formalin is known to penetrate tissue rapidly, but fix slowly. This phenomenon results from the chemical equilibrium of formaldehyde in aqueous solution being >99% methylene glycol and <0.1% reactive formaldehyde.[14] Buffered formaldehyde is inexpensive, readily available, does not interfere with histological staining, is non-coagulating and preserves native tissue architecture. Unlike ethanol, formalin does not cause excessive tissue shrinkage or distortion of cellular structure.[14] Despite its utility as a tissue preservative, it is important to note that formaldehyde has been classified by WHO as harmful to human health, so care must be taken to reduce exposure in research and clinical settings. Acute effects of formaldehyde exposure include eye and nasal irritation and formaldehyde has been classified as a carcinogen by the International Agency for Research on Cancer.[15]

The extraction and identification by mass spectrometry (MS) of proteins from FFPE tissues has been frustrated by formaldehyde-induced protein modifications formed during tissue fixation and histological processing. Studies of proteins and model peptides have identified three types of formaldehyde-induced chemical modifications: methylol (hydroxymethyl) adducts; Schiff's bases and stable methylene bridges (cross-links).[16,17] Methylol adducts and Schiff's bases are generally reversible; however, when present on primary amines or thiols, principally lysine and cysteine, they can undergo a second reaction with certain accessible amino acids and form cross-links. These partnering amino acids are arginine, asparagine, glutamine, histidine, tryptophan and tyrosine.[16,18] Protein cross-links have been identified in both model peptides[16] and whole proteins, such as insulin.[17] Additionally, the protein N-terminal amine can be converted to a stable 4-imidazolidione adduct[17] and a Mannich reaction can covalently join proximal tyrosine and arginine residues.[19]