Analytical and Bioanalytical Chemistry

, Volume 407, Issue 8, pp 2223–2243 | Cite as

The challenge of on-tissue digestion for MALDI MSI— a comparison of different protocols to improve imaging experiments

  • Hanna C. Diehl
  • Birte Beine
  • Julian Elm
  • Dennis Trede
  • Maike Ahrens
  • Martin Eisenacher
  • Katrin Marcus
  • Helmut E. Meyer
  • Corinna Henkel
Research Paper
Part of the following topical collections:
  1. Mass Spectrometry Imaging


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.

Graphical abstract

Example of experimental setup: Comparison of matrices DHB vs. HCCA (II) using FFPE tissue digested for 2 h. Overview of the statistic and structure analysis. (a) pLSA, only components with at least two clearly visible structures are displayed. (b) Mean of counted structures for all visible m/z values of theoretically digested MBP. The three numbers for each experimental condition are derived from counts of three different researchers (R1, R2, and R3). Color coding for (c) and (d): HCCA (II) in red and DHB in blue. (c) PCA of the mean spectra and (d) PCA of the spectra group


Matrix-assisted laser desorption/ionization (MALDI) Mass spectrometry imaging (MSI) Sample preparation Peptide imaging Formalin-fixed paraffin-embedded (FFPE) Tryptic digestion LysC-mix 

Supplementary material

216_2014_8345_MOESM1_ESM.pdf (1.5 mb)
ESM 1(PDF 1.53 mb)


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Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Hanna C. Diehl
    • 1
  • Birte Beine
    • 1
    • 2
  • Julian Elm
    • 1
  • Dennis Trede
    • 3
  • Maike Ahrens
    • 1
  • Martin Eisenacher
    • 1
  • Katrin Marcus
    • 1
  • Helmut E. Meyer
    • 2
  • Corinna Henkel
    • 1
    • 2
  1. 1.Medizinisches Proteom-CenterRuhr-University BochumBochumGermany
  2. 2.Leibniz-Institut für Analytische Wissenschaften – ISAS – e.V.DortmundGermany
  3. 3.SCiLS GmbHBremenGermany

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