Application of 2‐D DIGE to formalin‐fixed, paraffin‐embedded tissues

Abstract

The ability to investigate the proteome of formalin-fixed, paraffin-embedded (FFPE) tissues can be considered a major recent achievement in the field of clinical proteomics. However, gel-based approaches to the investigation of FFPE tissue proteomes have lagged behind, mainly because of insufficient quality of full-length protein extracts. Here, the 2-D DIGE technology was investigated for applicability to FFPE proteins, for internal reproducibility among replicate FFPE extracts, and for comparability between FFPE and fresh-frozen tissue profiles. The 2-D DIGE patterns obtained upon labeling and electrophoresis of replicate FFPE tissue extracts were highly reproducible, with satisfactory resolution and complexity. Moreover, the implementation of DIGE enabled to highlight and characterize the consistent differences found in the FFPE profiles compared with fresh-frozen profiles, represented by an acidic shift, directly correlated to the protein pI value, and by a reduction in spot signal intensity, directly correlated to molecular weight and percentage of lysine residues. Being constantly and reproducibly present in all FFPE tissue extract replicates at similar extents, these modifications do not appear to hinder the comparative analysis of FFPE tissue extracts by 2-D DIGE, opening the way to its application for the differential proteomic investigation of archival tissue repositories.

Proteomics and Pathway Analyses of the Milk Fat Globule in Sheep Naturally Infected by Mycoplasma agalactiae Provide Indications of the In Vivo Response of the Mammary Epithelium to Bacterial Infection

“…The labeled protein samples and the internal pooled standard were mixed in suitable combinations, brought to the final rehydration volume with IPG buffer (GE Healthcare) and Destreak rehydration solution (GE Healthcare), and applied to 24-cm IPG strips (pH 3 to 11, nonlinear; GE Healthcare) by passive rehydration overnight at room temperature (see Table S1 in the supplemental material). Rehydrated strips were then run together in an IPGphor device equipped with an Ettan IPGphor 3 loading manifold (GE Healthcare) at 20°C for a total of about 90,000 V-h. After isoelectric focusing (IEF), the strips were equilibrated (6), subjected to seconddimension SDS-PAGE, and digitalized as described previously (44). The images were analyzed with a DeCyder batch processor and differential in-gel analysis (DIA) modules (GE Healthcare).…”

“…After electrophoresis, the gel slab was subjected to colloidal Coomassie staining (8). Visible protein spots of interest were excised manually from the gels, and mass spectra were recorded on a matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) instrument (MALDI micro MX; Waters, Manchester, United Kingdom) as described previously (2,7,44). Raw data, reported as monoisotopic masses, were then introduced into our in-house Mascot peptide mass fingerprinting software (version 2.2; Matrix Science, Boston, MA) and used for protein identification.…”

Proteomic analysis of FFPE tissue: barriers to clinical impact

“…Studies of FFPE tissue protein extracts by 2-D difference gel electrophoresis exhibit high molecular weight complexes arising from residual crosslinked proteins. Relative to fresh tissue, FFPE gel profiles also exhibit an acid shift correlated to protein isoelectric point values and a reduction in spot intensity correlated to protein molecular weight and lysine content [15]. These results indicate the presence of residual formaldehyde adducts on lysine and possibly other amino acid residues.…”

Methacarn as a whole brain fixative for gene and protein expression analyses of specific brain regions in rats

“…In formalin-fixed tissue samples, reduction in spot signal intensity and acidic shift of extracted polypeptides usually occur in 2D-DIGE analysis (Tanca et al, 2011). Also, microdissected paraffin-embedded tissue samples stained with hematoxylin and eosin after fixation with 70% ethanol or acetone resulted in reduction of spot number of proteins in 2D-gel (De Souza et al, 2004).…”