Histology-Compatible MALDI Mass Spectrometry Based Imaging of Neuronal Lipids for Subsequent Immunofluorescent Staining

Abstract

Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) enables acquisition of spatial distribution maps for molecular species in situ. This can provide comprehensive insights on the pathophysiology of different diseases. However, current sample preparation and MALDI-IMS acquisition methods have limitations in preserving molecular and histological tissue morphology, resulting in interfered correspondence of MALDI-IMS data with subsequently acquired immunofluorescent staining results. We here investigated the histology compatibility of MALDI-IMS to image neuronal lipids in rodent brain tissue with subsequent immunohistochemistry and fluorescent staining of histological features. This was achieved by sublimation of a low ionization energy matrix compound, 1,5-diaminonapthalene (1,5-DAN), minimizing the number of low-energy laser shots. This yielded improved lipid spectral quality and speed of data acquisition and reduced matrix cluster formation along with preservation of specific histological information at cellular levels. This gentle, histology-compatible MALDI-IMS protocol also diminished thermal effects and mechanical stress created during nanosecond laser ablation processes that were prominent in subsequent immunofluorescent staining images but not with classical hematoxylin and eosin (H&E) staining on the same tissue section. Furthermore, this methodology proved to be a powerful strategy for investigating β-amyloid (Aβ) plaque-associated neuronal lipids as exemplified by performing high-resolution MALDI-IMS with subsequent fluorescent amyloid staining in a transgenic mouse model of Alzheimer's disease (tgSwe).

Mass spectrometry imaging of lipids: untargeted consensus spectra reveal spatial distributions in Niemann-Pick disease type C1

“…The mass spectrum for the on-tissue analysis (supplemental Fig. S1A-D) is provided for each replicate to evaluate m/z 612.3, a matrix ion of 1,5-diaminonapthalene (48). An example image of a matrix ion is provided to demonstrate reproducible matrix application (supplemental Fig.…”

Mass spectrometry is a multifaceted weapon to be used in the battle against Alzheimer's disease: Amyloid beta peptides and beyond

“…For example, matrices such 3‐aminoquinoline are very efficient for MALDI MS analysis of gangliosides in the negative‐ion mode, after a pre‐treatment with ethanol and ammonium formate to distinguish specific and different gangliosides patterns between AD and control brains . In any case, from the few examples reported above, it is clear that appropriate choice of matrix seems to be a critical step in the analysis of brain lipids with MALDI, as widely reported in the literature …”

“…179 In any case, from the few examples reported above, it is clear that appropriate choice of matrix seems to be a critical step in the analysis of brain lipids with MALDI, as widely reported in the literature. 180 FIGURE 1 Aβ (40 µM) modification by HNE (400 µM). Aβ (black).…”

Molecular imaging mass spectrometry for probing protein dynamics in neurodegenerative disease pathology

“…While this approach is well suited for IMS of lipids, comprehensive analysis of peptides and proteins is more challenging as desorption and ionization of protein and peptides require higher laser fluences leading to tissue distortion effects (Kaya et al . ). Therefore, a suitable matrix is required that is easy to ionize and requires lower laser energies.…”

“…Further, Kaya et al . () demonstrated a comprehensive imaging paradigm that employs a MALDI matrix that requires only low laser fluences for ionization and in turn facilitates histology compatible MALDI IMS of lipids as well as for subsequent fluorescent, immunohistological imaging on the same tissue sample. While this approach is well suited for IMS of lipids, comprehensive analysis of peptides and proteins is more challenging as desorption and ionization of protein and peptides require higher laser fluences leading to tissue distortion effects (Kaya et al .…”