Thermal Denaturation of Fresh Frozen Tissue Enhances\nMass Spectrometry Detection of Peptides

Thermal denaturation\n(TD), known as antigen retrieval, heats tissue\nsamples in a buffered solution to expose protein epitopes. Thermal\ndenaturation of formalin-fixed paraffin-embedded samples enhances\non-tissue tryptic digestion, increasing peptide detection using matrix-assisted\nlaser desorption ionization imaging mass spectrometry (MALDI IMS).\nWe investigated the tissue-dependent effects of TD on peptide MALDI\nIMS and liquid chromatography-tandem mass spectrometry signal in unfixed,\nfrozen human colon, ovary, and pancreas tissue. In a triplicate experiment\nusing time-of-flight, orbitrap, and Fourier-transform ion cyclotron\nresonance mass spectrometry platforms, we found that TD had a tissue-dependent\neffect on peptide signal, resulting in a (22.5%) improvement in peptide\ndetection from the colon, a (73.3%) improvement in ovary tissue, and\na (96.6%) improvement in pancreas tissue. Biochemical analysis of\nidentified peptides shows that TD facilitates identification of hydrophobic\npeptides.