Proteomic analysis of RCL2 paraffin-embedded tissues

Abstract

Histopathological diagnosis in most of the world's hospitals is based upon formalin-fixed and paraffin-embedded (FFPE) tissues. Although this standard fixation and embedding procedure keeps the tissue in excellent form for morphological and immunohistological analysis, FFPE is inappropriate for nucleic acids and protein studies. We investigated the potential value of RCL2, a new non-toxic fixative, for sparing proteins preserved in paraffin-embedded tissues. Normal colonic mucosa tissue was fixed in RCL2 prior to paraffin embedding (RCL2P), and then processed for quality and quantity of protein conservation, as compared to frozen and FFPE tissues using complementary proteomic analysis approaches. Using 4 different protein extraction protocols, RCL2P tissue consistently showed the highest protein yield. Similar protein patterns were observed with RCL2P and frozen tissues using mono and bi-dimensional electrophoresis. Moreover, membrane, cytoplasmic and nuclear proteins, as well as phosphorylated proteins, were successfully detected using western-blot. Furthermore, protein patterns observed by mass spectrometry analysis after laser-captured microdissection were found to be identical for frozen and RCL2-fixed tissues. At last, immunohistochemistry using various antibodies showed comparable results between both tissue storage methods. We concluded that RCL2 has great potential for performing both morphological and molecular analyses on the same archival paraffin-embedded tissue sample, and can be a new method for investigating protein biomarkers.

Fig 1

SDS-PAGE gels of extracted proteins from formalin and RCL2-fixed paraffin-embedded tissues compared to frozen colonic tissues. Proteins were extracted using protocols A (A), B (B), C (C) and Qproteome FFPE kit (D).

Fig 2

Representative silver stained two-dimensional electrophoresis maps obtained from FFPE tissues (panels A and B), RCL2P tissues (panels C and D), and frozen colonic mucosa tissues (panel E). Proteins were extracted using either protocol A (panels A, C, E) or protocol D (panels B and D). On panels C and E, the selected protein spots cut from the gels and analysed by MALDI TOF/TOF method were indicated.

Fig 3

MS analysis of spot 1 excised from 2-DE gels obtained from RCL2P and frozen tissues respectively were presented on panel A, and the two subsequent MS/MS analysis of the 1783.92 kD protein peak are shown on panel B. Via Swiss-Prot database searching the Heat Shock protein β-1 (accession number P04792, theoretical pl value 5.98 and theoretical Mr 22826 Da) was identified. Mascott scores obtained were 74 and 71 for protein extracted from RCL2P and Frozen maps, respectively and the sequence coverage were 54 and 76%. The two MS/MS spectra of m/z 1783.92 generated a partial y ion series which led to the identification via Swiss-Prot database search of VSLDVNHFAPDELTVK from Heat Shock protein beta-1.

Fig 4

Western blot performed using antibodies against E-cadherin, Sp1, α-tubulin, phospho MEK 1/2, phospho-p42 and phospho-p44 MAPK using protocol A extraction on two colonic mucosa tissue samples (A). Western blot performed using Sp1 and α-tubulin antibodies on proteins extracted using protocol D, compared to protocol A, of one colonic mucosa tissue sample (B).

Fig 5

Morphology and immunohistochemical staining of hMLH1, cytokeratin AE1/AE3 and E-cadherin of normal colonic mucosa fixed with formalin and RCL2 on the left and right panels, respectively. HES stained sections (A, B); nuclear hMLH1 (C, D); cytoplasmic cytokeratin AE1/AE3 (E, F); membrane E-cadherin (G, H) and cytoplasmic and nuclear phospho-MEK (I, J) immunoreactivities are presented. Original magnification x 400. For panels A and B, original magnification x100, insets x400.

Fig 6

Mass spectrometry analysis of proteins extracted from whole RCL2P compared with frozen normal colonic tissue (A). LCM and mass spectrometry analysis of proteins extracted from 500 and 1500 cells from RCL2-fixed and frozen tissues (B).