MALDI Mass Spectrometry Imaging: A Novel Tool for the Identification and Classification of Amyloidosis

Abstract

Amyloidosis is a group of diseases caused by extracellular accumulation of fibrillar polypeptide aggregates. So far, diagnosis is performed by Congo red staining of tissue sections in combination with polarization microscopy. Subsequent identification of the causative protein by immunohistochemistry harbors some difficulties regarding sensitivity and specificity. Mass spectrometry based approaches have been demonstrated to constitute a reliable method to supplement typing of amyloidosis, but still depend on Congo red staining. In the present study, we used matrix-assisted laser desorption/ionization mass spectrometry imaging coupled with ion mobility separation (MALDI-IMS MSI) to investigate amyloid deposits in formalin-fixed and paraffin-embedded tissue samples. Utilizing a novel peptide filter method, we found a universal peptide signature for amyloidoses. Furthermore, differences in the peptide composition of ALλ and ATTR amyloid were revealed and used to build a reliable classification model. Integrating the peptide filter in MALDI-IMS MSI analysis, we developed a bioinformatics workflow facilitating the identification and classification of amyloidosis in a less time and sample-consuming experimental setup. Our findings demonstrate also the feasibility to investigate the amyloid's protein composition, thus paving the way to establish classification models for the diverse types of amyloidoses and to shed further light on the complex process of amyloidogenesis.

Keywords: MALDI MS imaging; amyloidosis; formalin-fixed and paraffin-embedded; ion mobility separation.

Figure 1

Comparison of the three different mass spectrometry‐based approaches for the identification and classification of amyloidosis: A) Congo red staining followed by laser microdissection coupled with LC‐MS/MS, B) Congo red staining followed by parallel analysis of two consecutive tissue sections by LC‐MS/MS and MALDI MSI, C) MALDI MSI independent from Congo red staining as presented in this study. *Steps that require guidance of the Congo red staining.

Figure 2

Correlation between immunohistochemistry and MALDI‐IMS MSI analysis of six different amyloid types. Each row displays a panel consisting of immunostaining A), magnification B), images of peptide masses at m/z 968.55 (ApoE), 1314.68 (VTN), and 1811.89 (SAP) (C–E, respectively). Immunohistochemical staining shows a distribution pattern within the tissue sections similar to the areas with high signal intensities for the peptide masses. Characteristic tissue regions (green rectangle) are magnified for the immunohistochemical staining to visualize the specific enrichment and colocalization of the peptides in amyloid deposits. ATTR: transthyretin‐derived amyloidosis; AA: amyloid A‐derived amyloidosis; ALλ: immunoglobulin λ light chain derived amyloidosis; AFib: fibrinogen‐derived amyloidosis; AApoAI: apolipoprotein AI‐derived amyloidosis; ALys: lysozyme‐derived amyloidosis. The spatial resolution for the peptide images is 200 μm. Scale bar: 2 mM.

Figure 3

Overview of the 16 different cases with AA‐, AApoAI‐, AFib‐, ALys‐, ATTR‐, and ALλ amyloidosis, respectively, in FFPE tissue sections from brain (B), heart (H), kidney (K), liver (L), or spleen (S). For each case it is shown, which peptides of the common components ApoE, SAP, VTN, and the amyloidogenic proteins ApoAI, SAA, TTR were detected (green) or not detected (red). Peptides of ApoAI and SAA were also detected for noncorresponding amyloid cases (yellow). The detection frequency of each peptide mass was determined by dividing the number of amyloid cases with detection (n _D) by the number of all amyloid cases (n _A).