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

Effect of Pre-analytical Factors on Proteomic Profiling

Changes in protein levels, either through protein degradation or continued protein expression, can occur in the interval between tissue excision and placement of the specimen in formalin or freezing medium. This duration before tissue stabilization is often referred to as the cold ischemia interval.[20] In one of the few studies of cold ischemia, Thompson et al. recommended that tissue be stored on ice immediately following excision and placed in formalin within 30 min. However, in normal clinical practice biopsy specimens are typically stored at 4°C and fixed within 1 h. Delays in fixation or freezing can lead to a reduction in high molecular weight proteins with a concomitant increase in low molecular weight proteins,[21] suggesting the occurrence of proteolysis. The magnitude of this change is tissue dependent and occurs most rapidly in neural tissue where protein degradation begins within 1 min after excision.[22] Phosphoproteins are particularly susceptible and their levels can drop or increase by up to 20% within 20 min of excision, leading some to suggest that FFPE tissue should not be used to evaluate the phosphorylation state of proteins.[20,23,24] The size of tissue samples can affect proteomic analysis as proteins continue to degrade at the core of large specimens after emersion in formalin.[14] The majority of studies have concluded that an increase in fixation time beyond 24 h reduces the amount of extractable protein[25,26] and lowers the number of spectral counts, the number of peptides detected and the number of proteins identified by mass spectrometric analysis of the tissue extract. In one example, Wolff et al. found a 20% reduction in extractable proteins from lymph node FFPE tissue when the fixation time was extended from 24 to 48 h.[25]

The effect of tissue processing on FFPE tissue proteomics has received little attention. Xie et al. showed that when tissues were inadequately dehydrated during processing, the retained water resulted in reduced antigenicity and that exposure to high humidity resulted in protein degradation during storage.[27] Protein integrity was improved by storing FFPE tissues at low temperatures (4°C). The effect of storage time on the extraction efficiency and analysis of FFPE tissue has been studied by several groups. Xie et al. concluded that oxidation did not appear to contribute to protein degradation in FFPE tissues;[27] however, Sprung et al. found that methionine oxidation in archival FFPE tissue increased from 17% after 1 year to 25% after 10 years of storage. They also observed a decreased representation of proteins with C-terminal lysine residues as storage time increased.[28] Scicchitano compared protein recovery from matched liver tissue prepared as optimal cutting temperature media (OCT)-frozen specimens versus FFPE tissue blocks and FFPE slides.[29] Recovery of proteins by laser-capture microdissection (LCM) of liver samples isolated from FFPE slides was close to 90% compared with OCT-frozen tissue. For whole liver tissue sections, protein recovery dropped to approximately 42% with greater variability between specimens. Scicchitano observed that no intact proteins >120 kDa were detected by sodium dodecyl sulfate (SDS)-PAGE and western blot, suggesting that hydrolysis of the peptide backbone may occur in FFPE tissue.[29] Similar findings were reported by Fowler et al..[30]

Balgley et al. investigated the effect of archival storage time on the proteomic analysis of nine uterine leiomyomas dating from 1990 to 2002 using transient capillary isotachophoresis/capillary zone electrophoresis-coupled linear ion-trap mass spectrometry.[31] Evaluation of the results by the Pearson coefficient indicated that the leiomyoma specimens correlated strongly with each other, but not by archival age. Analysis of the MS data by k-means clustering similarly failed to reveal a strong effect of archival age on the proteome. However, when individual proteins were analyzed, k-means clustering of the results suggested that low abundance proteins are more difficult to retrieve as the tissue blocks aged beyond 10 years. A shotgun proteomic analysis of colon adenoma FFPE tissues carried out by Sprung et al. found that the proteome did not change significantly for archival storage times of up to 10 years.[28] In contrast to these findings, Wolff et al. found that protein recovery from FFPE colon tissue decreased with age, with 42% less protein recoverable from specimens dating from 1990 relative to specimens from 2010.[25]

There is currently no clear consensus on the effect of pre-analytical factors on the proteome recovered from FFPE tissues. However, recent studies do appear to indicate that archival storage time has a smaller impact on the FFPE proteome than excessive formalin fixation times, particularly for fixation that extends beyond 24 h. Although some of the studies mentioned above may appear contradictory, this more likely reflects variations in tissue handling compounded by our incomplete understanding of the interdependencies of pre-analytical factors on FFPE proteomic outcomes.

A cautionary note is warranted regarding studies that estimate the success of protein extraction from FFPE tissues by comparing the results with those from matched fresh or frozen tissue. Such conclusions are only valid for averages from multiple replicates because even for matched fresh tissue specimens, overlap in the identified proteins can be as low as 50% due to variations in sample preparation and undersampling of the resulting peptides.[31]