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Abstract
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 (LC-MS/MS) 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.
Supplementary materials
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Supplemental Materials
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Table S2. PTXQC missed cleavage report.
Figure S1. Amino acid composition of LC-MS identified peptides from microextraction samples
Figure S2-4. Average ultrafleXtreme MALDI IMS spectra from colon, ovary, and pancreas tissue.
Figure S5-7. ultrafleXtreme MALDI IMS ion images and per-pixel intensities in colon, ovary, and pancreas tissue.
Figure S8. FTICR MALDI IMS spectra
Figure S9. Post-MALDI IMS H&E stains, ion images, and per-pixel intensities in pancreas tissue.
Figure S10. Comparison of pancreas tissue adherence using an indium tin oxide (ITO) slide compared to a poly-lysine coated ITO slide.
Figure S11. MALDI IMS ion images from pancreas tissue.
Figure S12. Average IMS spectra of region directly outside of colon, ovary, and pancreas tissues.
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Table S1
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Table with identified and differentially abundant proteins in LC-MS data
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Table S3
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List of MALDI features above a 5% intensity threshold in colon IMS with indication of which ions are detected in TD, NTD, or both tissue types along with every unthresholded feature in TD colon and NTD colon.
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Table S4
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List of MALDI features above a 5% intensity threshold in ovary IMS with indication of which ions are detected in TD, NTD, or both tissue types along with every unthresholded feature in TD ovary and NTD ovary.
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Table S5
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List of MALDI features above a 5% intensity threshold in pancreas IMS with indication of which ions are detected in TD, NTD, or both tissue types along with every unthresholded feature in TD pancreas and NTD pancreas.
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