Abstract
Mass spectrometry imaging (MSI) has become a powerful and successful tool in the context of biomarker detection especially in recent years. This emerging technique is based on the combination of histological information of a tissue and its corresponding spatial resolved mass spectrometric information. The identification of differentially expressed protein peaks between samples is still the method’s bottleneck. Therefore, peptide MSI compared to protein MSI is closer to the final goal of identification since peptides are easier to measure than proteins. Nevertheless, the processing of peptide imaging samples is challenging due to experimental complexity. To address this issue, a method development study for peptide MSI using cryoconserved and formalin-fixed paraffin-embedded (FFPE) rat brain tissue is provided. Different digestion times, matrices, and proteases were tested to define an optimal workflow for peptide MSI. All practical experiments were done in triplicates and analyzed by the SCiLS Lab software, using structures derived from myelin basic protein (MBP) peaks, principal component analysis (PCA) and probabilistic latent semantic analysis (pLSA) to rate the experiments’ quality. Blinded experimental evaluation in case of defining countable structures in the datasets was performed by three individuals. Such an extensive method development for peptide matrix-assisted laser desorption/ionization (MALDI) imaging experiments has not been performed so far, and the resulting problems and consequences were analyzed and discussed.
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Acknowledgments
We would like to thank Judith Baronner for her skilful technical assistance. We thank Klaus Steinhorst for excellent support dealing with the SCiLS Lab. We thank Promega Corporation for providing test samples of the LysC-mix. Additionally, we thank Michael Becker for general MALDI imaging support. We thank Mark Proctor for proof reading. HD, BB, MA, ME, and CH were financially supported by PURE (Protein research Unit Ruhr within Europe).
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The authors declare that there is no conflict of interest regarding the publication of this article.
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Published in the topical collection Mass Spectrometry Imaging with guest editors Andreas Römpp and Uwe Karst.
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Diehl, H.C., Beine, B., Elm, J. et al. The challenge of on-tissue digestion for MALDI MSI— a comparison of different protocols to improve imaging experiments. Anal Bioanal Chem 407, 2223–2243 (2015). https://doi.org/10.1007/s00216-014-8345-z
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DOI: https://doi.org/10.1007/s00216-014-8345-z