Background Compared to histology and immunohistochemistry, MALDI-MSI can localize many molecules in a single tissue section. However, the spatial resolution falls short of light microscopy due to instrument and sample preparation limitations. In particular, trypsin digestion required for MALDI-MSI of peptides from FFPE samples is a major source of analyte delocalization. Using a novel sample preparation method, it is possible to distinguish fine structures using MALDI-MSI. Spatial resolution quality was assessed using different FFPE tissues: mouse jejunum, and a human teratoma sample. Design FFPE sections (5 μm) were subjected to deparaffinization, rehydration and heat-induced epitope retrieval. Samples were sprayed with 0.025 µg/µl trypsin 0.05% Glycerol and digested at 50°C using saturated K2SO44 solution to maintain 97% humidity. After coating with alpha-cyano-4-hydroxycinnamic acid matrix, samples were measured at 50 µm step size with a MALDI-TOF mass spectrometer. Following acquisition, the tissues were stained with HE, annotated by a pathologist, and co-registered to the MALDI-MSI datasets. Results Mouse jejunum was used to assess analyte delocalization during the method development and evaluated for mass spectral quality and spatial homogeneity. The method was optimized to yield informative mass spectra while maintaining the spatial delocalization of analytes, two usually contradictory goals. The approach to control the digest conditions based on the deliquescence of K2SO44 allowed both goals by enabling consistent results. The various anatomical regions of the teratoma could be differentiated based on their mass spectrometric profiles and at high spatial resolution, as was evident by several anatomical structures. Most telling was a mass signal that could be attributed to the corneal layer of the skin. Conclusion Spatial resolution of MALDI-MSI from FFPE samples has been improved to discrete structural features, and has been demonstrated on different tissues.
Improved spatial resolution of FFPE MALDI tissuetyping
Casadonte R;
2018-01-01
Abstract
Background Compared to histology and immunohistochemistry, MALDI-MSI can localize many molecules in a single tissue section. However, the spatial resolution falls short of light microscopy due to instrument and sample preparation limitations. In particular, trypsin digestion required for MALDI-MSI of peptides from FFPE samples is a major source of analyte delocalization. Using a novel sample preparation method, it is possible to distinguish fine structures using MALDI-MSI. Spatial resolution quality was assessed using different FFPE tissues: mouse jejunum, and a human teratoma sample. Design FFPE sections (5 μm) were subjected to deparaffinization, rehydration and heat-induced epitope retrieval. Samples were sprayed with 0.025 µg/µl trypsin 0.05% Glycerol and digested at 50°C using saturated K2SO44 solution to maintain 97% humidity. After coating with alpha-cyano-4-hydroxycinnamic acid matrix, samples were measured at 50 µm step size with a MALDI-TOF mass spectrometer. Following acquisition, the tissues were stained with HE, annotated by a pathologist, and co-registered to the MALDI-MSI datasets. Results Mouse jejunum was used to assess analyte delocalization during the method development and evaluated for mass spectral quality and spatial homogeneity. The method was optimized to yield informative mass spectra while maintaining the spatial delocalization of analytes, two usually contradictory goals. The approach to control the digest conditions based on the deliquescence of K2SO44 allowed both goals by enabling consistent results. The various anatomical regions of the teratoma could be differentiated based on their mass spectrometric profiles and at high spatial resolution, as was evident by several anatomical structures. Most telling was a mass signal that could be attributed to the corneal layer of the skin. Conclusion Spatial resolution of MALDI-MSI from FFPE samples has been improved to discrete structural features, and has been demonstrated on different tissues.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
