MALDI Imaging of Formalin-Fixed Paraffin-Embedded Tissues: Application to Model Animals of Parkinson Disease for Biomarker Hunting

Abstract

A common technique for the long-term storage of tissues in hospitals and clinical laboratories is preservation in formalin-fixed paraffin-embedded (FFPE) blocks. Such tissues stored for more than five years have not been useful for proteomic studies focused on biomarker discovery. Recently, MS-based proteomic analyses of FFPE showed positive results on blocks stored for less than 2 days. However, most samples are stored for more than one year, and thus our objective was to establish a novel strategy using as a model system 6-hydroxydopamine (6-OHDA) treated rat brain tissues stored in FFPE blocks for more than 9 years. We examined MALDI tissue profiling combining the use of automatic spotting of the MALDI matrix with in situ tissue enzymatic digestion. On adjacent sections, the identification of compounds is carried out by tissue digestion followed by nanoLC/MS-MS analysis. The combination of these approaches provides MALDI direct analysis, MALDI/MS imaging, as well as the localization of a large number of proteins. This method is validated since the analyses confirmed that ubiquitin, trans-elongation factor 1, hexokinase, and the Neurofilament M are down-regulated as previously shown in human or Parkinson animal models. In contrast, peroxidoredoxin 6, F1 ATPase, and alpha-enolase are up-regulated. In addition, we uncovered three novel putative biomarkers, the trans-elongation factor 1 (eEF1) and the collapsin response mediator 1 and 2 from protein libraries. Finally, we validate the CRMP-2 protein using immunocytochemistry and MALDI imaging based on the different ions from trypsic digestion of the protein. The access to archived FFPE tissue using MALDI profiling and imaging opens a whole new area in clinical studies and biomarker discovery from hospital biopsy libraries.

MALDI In-Source Decay, from Sequencing to Imaging

“…In most published applications, proteins are targeted as potential biomarkers of pathology, as in many studies, for ovarian tumors [80,81], renal carcinoma [82], or Parkinson's disease [83,84]. For small molecules analysis, two main classes may be distinguished -exogenous drugs and endogenous metabolites.…”

The C-terminal fragment of the immunoproteasome PA28S (Reg alpha) as an early diagnosis and tumor-relapse biomarker: evidence from mass spectrometry profiling

“…A decade of developments in instrumentation and chemistry has been required to achieve optimal extraction, detection, and spatial resolution when mapping compounds at the tissue level (Franck et al 2009a, b;Lemaire et al 2006a, b;2007a;van Remoortere et al 2010). These developments, combined with using the appropriate data-processing tools, gave rise to the discovery of biomarkers for diverse pathologies (Bonnel et al 2011;Djidja et al 2010;McCombie et al 2005;Stauber et al 2008), of which cancer is the most studied. MALDI mass spectrometry imaging (MALDI-MSI) technology makes it possible to study the molecular profiles of the benign and malignant portions of a solid tumor.…”

Multivariate analyses for biomarkers hunting and validation through on-tissue bottom-up or in-source decay in MALDI-MSI: application to prostate cancer

“…This is explained principally based on two factors: it is difficult to use a proper top-down analysis with ion selection on MALDI instruments, and archived clinical samples (FFPE) cannot be used for MSI without a digestion step [35,37,38]. Therefore, marker screening from a classical MSI sequence is performed.…”

“…Consequently, many efforts by others and by our group [35,37] have been implemented to find a strategy to analyze tissues using MALDI-MSI [37,[48][49][50]. Most of the libraries are builds of FFPE tissues because this preservation protocol produces well-conserved morphology and high stability along time avoiding degradation of molecules.…”