Background MALDI imaging is a label-free analytical technique for direct analysis of samples that revolutionized biological mass spectrometry, and especially proteomics, as ionized biomolecules maintain their spatial integrity. For large tissue sections and/or tissue microarrays (~4 cm2), MALDI imaging analysis at high spatial resolution was previously impaired by the prohibitively slow acquisition speed of existing platforms. In this study, we present the application of a novel MALDI instrument for the analysis of tryptic peptides from FFPE tissue sections. In addition to allowing acquisition speed of >20 pixel/second, the newly developed system allows quasi-square, discrete pixels and continuous stage movement, contributing to consistent data quality even at small pixel size. Methods FFPE kidney and lung tumor tissues were subjected to deparaffination and heat-induced epitope retrieval. Trypsin solution was sprayed on the tissue sections using an automatic spray instrument. After 1.5 h digestion, alpha-cyano-4-hydroxycinnamic acid matrix was deposited using the same sprayer device and the tissues were analyzed for peptides with an AutoflexSpeed MALDI TOF/TOF mass spectrometer and a novel rapifleX MALDI TissueTyper (Bruker Daltonik GmbH, Bremen, Germany). Ion images were visualized using flexImaging software at 50 μm spatial resolution. Results MALDI data generated from the two platforms (AutoflexSpeed and rapifleX TissueTyper) were evaluated and compared based on the acquisition speed, time, and maintenance of the tissue integrity. Analyses of multiple large tissue sections were analysed with the novel high-speed instrument, enabling extremely fast acquisition speeds (~50 pixel/sec). A single tissue section allowed a measurement of 160,694 spectra (pixels) at 50μm pixel size with a total acquisition time of ~1.5 h. Similar manageable timeframe was used for the analysis of a lung cancer tissue microarray including 99 tissue cores, that generated 51,932 spectra data. Conclusions A novel MALDI imaging platform has been used for the direct analysis of large FFPE tissue sections with high-speed (up to 50 pixel/sec), high resolution, and high throughput. The statistical analysis of spectral data from several hundred thousand pixels represents another challenge which we have addressed using new software architecture.
A Novel MALDI Imaging Technology for High-Speed Analysis of FFPE Tissues
Casadonte R;
2016-01-01
Abstract
Background MALDI imaging is a label-free analytical technique for direct analysis of samples that revolutionized biological mass spectrometry, and especially proteomics, as ionized biomolecules maintain their spatial integrity. For large tissue sections and/or tissue microarrays (~4 cm2), MALDI imaging analysis at high spatial resolution was previously impaired by the prohibitively slow acquisition speed of existing platforms. In this study, we present the application of a novel MALDI instrument for the analysis of tryptic peptides from FFPE tissue sections. In addition to allowing acquisition speed of >20 pixel/second, the newly developed system allows quasi-square, discrete pixels and continuous stage movement, contributing to consistent data quality even at small pixel size. Methods FFPE kidney and lung tumor tissues were subjected to deparaffination and heat-induced epitope retrieval. Trypsin solution was sprayed on the tissue sections using an automatic spray instrument. After 1.5 h digestion, alpha-cyano-4-hydroxycinnamic acid matrix was deposited using the same sprayer device and the tissues were analyzed for peptides with an AutoflexSpeed MALDI TOF/TOF mass spectrometer and a novel rapifleX MALDI TissueTyper (Bruker Daltonik GmbH, Bremen, Germany). Ion images were visualized using flexImaging software at 50 μm spatial resolution. Results MALDI data generated from the two platforms (AutoflexSpeed and rapifleX TissueTyper) were evaluated and compared based on the acquisition speed, time, and maintenance of the tissue integrity. Analyses of multiple large tissue sections were analysed with the novel high-speed instrument, enabling extremely fast acquisition speeds (~50 pixel/sec). A single tissue section allowed a measurement of 160,694 spectra (pixels) at 50μm pixel size with a total acquisition time of ~1.5 h. Similar manageable timeframe was used for the analysis of a lung cancer tissue microarray including 99 tissue cores, that generated 51,932 spectra data. Conclusions A novel MALDI imaging platform has been used for the direct analysis of large FFPE tissue sections with high-speed (up to 50 pixel/sec), high resolution, and high throughput. The statistical analysis of spectral data from several hundred thousand pixels represents another challenge which we have addressed using new software architecture.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
