Application of Mass Spectrometry for Identification of Biomarkers in Formaldehyde-Fixed Paraffin Embedded Specimens

Mass Spectrometry (MS) has recently been utilized for the identification of biomarkers in peripheral blood and in urine, whereas its application in tissue samples remains limited. In this review article, we introduce a novel application of MS using archived Formalin-Fixed Paraffin-Embedded (FFPE) tissue. This method addresses the significant technical challenges for protein ionization, including amino acid residue modification of proteins. While various immunochemical pretreatments for enhancing the ionization signal of peptides have been reported, significant developments have yet to be achieved. We performed a simplified chemical pretreatment method for preparing tissue sections involving heating in acetonitrile-containing buffer under airtight and pressurized conditions. Analysis revealed that the number and intensity of ionized peptide peaks obtained from pretreated tissue were significantly higher than from untreated (control) tissue. This highly sensitive treatment may enable MALDI-MS and LC/MS (liquid chromatography/MS) analyses of archived pathological FFPE samples in the hospital, leading to the identification of new biomarkers. instrument to capture defined areas. First, the subjective lesion in the FFPE tissue (10 mm 2 in total) was collected into a 500-µL well. Each micro dissected sample was suspended in 20 µL of 0.1 mol/L NH 4 HCO 3 containing 30% (v/v) CH 3 CN in the wells, and then centrifuged at 10,000 x g for 1 min. Tubes were heated at 95°C for 90 min. After cooling, the tissues were digested with trypsin at 37°C overnight, and then heated to 95°C for 5 min for enzyme deactivation. After drying, samples were resuspended in 0.1% trifluoroacetic acid with 2% CH 3 CN and the final protein concentration was adjusted to 0.2 µg/µL. Un expectedly, 1.0×10 4 peptides and 1.0×10 2–3 proteins were identified in the various formaldehyde FFPE tissues including lung, synovium, pancreas, and brain; similar numbers were obtained with frozen samples. Most of the detected proteins were cytoskeletal proteins, including beta-tubulin 3, immunoglobulin, and vimentin.