An approach to optimize sample preparation for MALDI imaging MS of FFPE sections using fractional factorial design of experiments

Abstract

A standardized workflow for matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI imaging MS) is a prerequisite for the routine use of this promising technology in clinical applications. We present an approach to develop standard operating procedures for MALDI imaging MS sample preparation of formalin-fixed and paraffin-embedded (FFPE) tissue sections based on a novel quantitative measure of dataset quality. To cover many parts of the complex workflow and simultaneously test several parameters, experiments were planned according to a fractional factorial design of experiments (DoE). The effect of ten different experiment parameters was investigated in two distinct DoE sets, each consisting of eight experiments. FFPE rat brain sections were used as standard material because of low biological variance. The mean peak intensity and a recently proposed spatial complexity measure were calculated for a list of 26 predefined peptides obtained by in silico digestion of five different proteins and served as quality criteria. A five-way analysis of variance (ANOVA) was applied on the final scores to retrieve a ranking of experiment parameters with increasing impact on data variance.

MALDI imaging experiments were planned according to fractional factorial design of experiments for the parameters under study. Selected peptide images were evaluated by the chosen quality metric (structure and intensity for a given peak list), and the calculated values were used as an input for the ANOVA. The parameters with the highest impact on the quality were deduced and SOPs recommended.

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Acknowledgments

We would like to thank Olga Vitek for valuable discussions regarding the statistical design of experiments. The authors gratefully acknowledge the financial support from the European Commission Seventh Framework Program (project 3D-MASSOMICS, grant 305259), the German Science Foundation (DFG core facility MALDI-MULTI), and the German Central Innovation Program for SMEs of the German Federal Ministry of Economic Affairs and Energy (BMWI-ZIM grant 2443904SB4).

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Correspondence to Janina Oetjen.

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Conflict of interest

Theodore Alexandrov is the Scientific Director, Peter Maass is on the Advisory Board, and Tobias Boskamp is a consultant for SCiLS GmbH, a company that develops and markets software for imaging mass spectrometry. At the time of analysis, Michael Becker was employed at Bruker Daltonics GmbH, a vendor of mass spectrometry instrumentation. All other authors declare no conflict of interest.

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This article does not contain any studies with human participants. All animal care guidelines according to the German animal protection law were met.

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Oetjen, J., Lachmund, D., Palmer, A. et al. An approach to optimize sample preparation for MALDI imaging MS of FFPE sections using fractional factorial design of experiments. Anal Bioanal Chem 408, 6729–6740 (2016). https://doi.org/10.1007/s00216-016-9793-4

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Keywords

  • MALDI imaging MS
  • Fractional design of experiments
  • SOP