Background
Amyloidosis is a disease caused by amyloid fibril protein accumulation, which can affect one or multiple organs, potentially leading to organ failure. Diagnosing amyloidosis is difficult due to its many subtypes, including AA, AL, and ATTR amyloidosis, each with varying symptoms. Current diagnosis involves Congo red staining, but it has limitations in quantification and specificity. A novel method called iprm-PASEF exploits MALDI imaging and offers a faster, spatially resolved, antibody-independent technique for identifying peptides while preserving tissue structure. In this study, we applied this tool to further evaluate its applicability on amyloidosis.
Method
FFPE slides of an Amyloidosis TMA including biopsies of 18 amyloidosis positive tissues were deparaffinized and antigen-retrieved before tryptic on-tissue digestion and matrix application. First, an initial MALDI TIMS MS1 survey measurement was performed, followed by the manual generation of a precursor list containing mass-to-charge ratios and ion mobility windows. In a second iprm-PASEF measurement, the selected precursors separated according to their ion mobility, and analyzed using multiplexed MALDI MS/MS imaging. Finally, peptide identification was conducted through peptide-to-spectrum matching using MASCOT.
Results
During this study, we characterized an amyloidosis TMA consisting of AA, AL and ATTR amyloidosis diseased tissue with MALDI imaging of tryptic peptides. We successfully identified eight amyloidosis related peptides derived from Serum Amyloid A, Vitronectin, Apolipoprotein E, Serum Amyloid P and Transthyretin Receptor in one single iprm-PASEF measurement. By iprm-PASEF, peptide identifications of all seven peptides were corroborated by MS/MS fragmentation simultaneously and in a spatial manner. All peptide signals precisely mapped to amyloidogenic plaques determined in a Congo red staining. Remarkably, one of these peptides even allowed for the subtyping of ATTR and AL amyloidosis.
Conclusion
We successfully characterized an amyloidosis TMA using iprm-PASEF-enabled MALDI imaging. This represents a significant step towards integrating MALDI imaging into the diagnostic process for amyloidosis.