Hidden treasure: Extended workflow yields thousand of proteins from archived FFPE tissue

Tissue storage

Stained sections of representative normal kidney cortex (upper panel) and conventional (clear cell) renal cell carcinoma (lower panel)

In the search for biomarkers of disease using proteomics techniques, many samples need to be analysed in order to validate the results and confirm the special significance of particular peptides or proteins. Apart from the common biological fluids like plasma and urine, tissue is another highly prized source with the advantage that many hospitals store archived tissue along with the relevant patient histories.

Tissue can be stored by freezing but for long-term storage it is better fixed in formalin then embedded in paraffin. It has been shown that these FFPE tissues are equivalent to fresh or frozen tissues once they have been rehydrated and fixation has been reversed by heating, so there is a ready supply in the hunt for biomarkers.

There have been many reports in which FFPE tissue has been used in proteomics studies guided by mass spectrometry but some researchers argue that they have not reached their potential because the number of proteins identified is in the low thousands at best, which is only about 20% of the entire proteome. One reason given for this shortcoming is the way in which the proteins are extracted, digested and prepared for analysis.

Now, three European scientists have fine tuned the workflow for microdissected FFPE tissue that allowed the detection of nearly 10,000 proteins. Jacek Wisniewski, Kamila Dus and Matthias Mann from the Max-Planck-Institute of Biochemistry, Martinsried, Germany and Wroclaw Medical University, Poland, used tissue from colonic adenomas to demonstrate their system.

Filter-aided sample preparation

The key principles in the novel sample preparation scheme were repeated fractionation of the sample combined with a modified version of filter-aided sample preparation (FASP). In FASP, the tissue sample is digested by an enzyme on a filter, in this case a spin ultrafiltration unit with a molecular weight cutoff of 30,000 Da. The method has several advantages over normal digestion by providing digests that are free from nucleic acids and other cell components but which maintain high sample concentrations due to the absence of a precipitation step.

In this case, samples of lysed adenoma tissue were subjected to FASP with two enzymes for successive digestions, using endoproteinase Lys-C followed by trypsin. After each digestion, the resulting peptides were released by centrifugation and subjected to strong anion exchange (SAX) chromatography to give four fractions (at pH 2, 4, 6, 11) from the Lys-C peptides and two fractions (pH 2, 5) from the tryptic peptides. After desalting, the peptides were analysed by LC-tandem MS using a long HPLC gradient on a reversed-phase column and a high-resolution mass spectrometer.

The proteins were identified by searching against a human protein database and the amounts of each protein were estimated by label-free quantitation by comparing the individual intensities to the total mass spectrometric signal of the proteome.

Thousands of proteins

A minimum amount of sample was needed to generate data that extended the proteome beyond its most abundant components. In preliminary tests on HeLa cells, 6 µg of peptides, corresponding to about 100,000 cells, was sufficient to lead to the identification of 8500 proteins. For adenoma tissue, 6 µg of peptides corresponded to about 100 µg of microdissected tissue.

The small sample size did not detract from the number of peptides identified following the extensive digestion and fractionation steps. Between 6000 and 18,000 peptides were identified in each of the six SAX fractions by the high-resolution mass spectrometric procedure, nearly 40% of the MS/MS scans allowing the unambiguous identification of a peptide.

An average of 55,144 unique peptides per run were identified from the six fractions, leading to the identification of an average of 9500 proteins, of which 98% were within a 10,000-fold expression range of abundance. This extensive analysis would allow a systematic analysis of the development of the adenoma cells and could be used to discover new biomarkers of the disease and follow its progression through different stages.

Long-term storage of FFPE tissue

So, the sample preparation regime provides an extremely detailed picture of the proteome using FFPE tissue. But one question still exists. For tissues that have been stored for several years, does the storage time affect the proteins in the tissue?

This question has been addressed by Rosamonde Banks and colleagues from St. James's University Hospital, Leeds, UK who undertook proteomic analyses of FFPE blocks of normal kidney tissues and clear cell renal carcinoma tissues that had been stored for long periods. Microdissected samples were digested by single-enzyme FASP for analysis by LC-tandem-MS on a high resolution mass spectrometer.

The study showed that the storage time had no significant effect on the number of proteins and their abundances for up to 10 years, which vindicates their use in biomarker discovery and the study of diseases.