Dataset Information

Site-to-Site Reproducibility and Spatial Resolution in MALDI-MSI of Peptides from Formalin-Fixed Paraffin-Embedded Samples.

ABSTRACT:

Purpose

To facilitate the transition of MALDI-MS Imaging (MALDI-MSI) from basic science to clinical application, it is necessary to analyze formalin-fixed paraffin-embedded (FFPE) tissues. The aim is to improve in situ tryptic digestion for MALDI-MSI of FFPE samples and determine if similar results would be reproducible if obtained from different sites.

Experimental design

FFPE tissues (mouse intestine, human ovarian teratoma, tissue microarray of tumor entities sampled from three different sites) are prepared for MALDI-MSI. Samples are coated with trypsin using an automated sprayer then incubated using deliquescence to maintain a stable humid environment. After digestion, samples are sprayed with CHCA using the same spraying device and analyzed with a rapifleX MALDI Tissuetyper at 50 µm spatial resolution. Data are analyzed using flexImaging, SCiLS, and R.

Results

Trypsin application and digestion are identified as sources of variation and loss of spatial resolution in the MALDI-MSI of FFPE samples. Using the described workflow, it is possible to discriminate discrete histological features in different tissues and enabled different sites to generate images of similar quality when assessed by spatial segmentation and PCA.

Conclusions and clinical relevance

Spatial resolution and site-to-site reproducibility can be maintained by adhering to a standardized MALDI-MSI workflow.

SUBMITTER: Ly A

PROVIDER: S-EPMC6590241 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

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Publications

Site-to-Site Reproducibility and Spatial Resolution in MALDI-MSI of Peptides from Formalin-Fixed Paraffin-Embedded Samples.

Ly Alice A Longuespée Rémi R Casadonte Rita R Wandernoth Petra P Schwamborn Kristina K Bollwein Christine C Marsching Christian C Kriegsmann Katharina K Hopf Carsten C Weichert Wilko W Kriegsmann Jörg J Schirmacher Peter P Kriegsmann Mark M Deininger Sören-Oliver SO

Proteomics. Clinical applications 20190104 1

<h4>Purpose</h4>To facilitate the transition of MALDI-MS Imaging (MALDI-MSI) from basic science to clinical application, it is necessary to analyze formalin-fixed paraffin-embedded (FFPE) tissues. The aim is to improve in situ tryptic digestion for MALDI-MSI of FFPE samples and determine if similar results would be reproducible if obtained from different sites.<h4>Experimental design</h4>FFPE tissues (mouse intestine, human ovarian teratoma, tissue microarray of tumor entities sampled from three ...[more]

PMID: 30408343

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Project description:BackgroundWe have developed a gene expression assay (Whole-Genome DASL), capable of generating whole-genome gene expression profiles from degraded samples such as formalin-fixed, paraffin-embedded (FFPE) specimens.Methodology/principal findingsWe demonstrated a similar level of sensitivity in gene detection between matched fresh-frozen (FF) and FFPE samples, with the number and overlap of probes detected in the FFPE samples being approximately 88% and 95% of that in the corresponding FF samples, respectively; 74% of the differentially expressed probes overlapped between the FF and FFPE pairs. The WG-DASL assay is also able to detect 1.3-1.5 and 1.5-2 -fold changes in intact and FFPE samples, respectively. The dynamic range for the assay is approximately 3 logs. Comparing the WG-DASL assay with an in vitro transcription-based labeling method yielded fold-change correlations of R(2) approximately 0.83, while fold-change comparisons with quantitative RT-PCR assays yielded R(2) approximately 0.86 and R(2) approximately 0.55 for intact and FFPE samples, respectively. Additionally, the WG-DASL assay yielded high self-correlations (R(2)>0.98) with low intact RNA inputs ranging from 1 ng to 100 ng; reproducible expression profiles were also obtained with 250 pg total RNA (R(2) approximately 0.92), with approximately 71% of the probes detected in 100 ng total RNA also detected at the 250 pg level. When FFPE samples were assayed, 1 ng total RNA yielded self-correlations of R(2) approximately 0.80, while still maintaining a correlation of R(2) approximately 0.75 with standard FFPE inputs (200 ng).Conclusions/significanceTaken together, these results show that WG-DASL assay provides a reliable platform for genome-wide expression profiling in archived materials. It also possesses utility within clinical settings where only limited quantities of samples may be available (e.g. microdissected material) or when minimally invasive procedures are performed (e.g. biopsied specimens).

Dataset Information

Site-to-Site Reproducibility and Spatial Resolution in MALDI-MSI of Peptides from Formalin-Fixed Paraffin-Embedded Samples.

Purpose

Experimental design

Results

Conclusions and clinical relevance

Publications

Site-to-Site Reproducibility and Spatial Resolution in MALDI-MSI of Peptides from Formalin-Fixed Paraffin-Embedded Samples.

Similar Datasets

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