Thermal Denaturation of Fresh Frozen Tissue Enhances Mass Spectrometry Detection of Peptides

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