MALDI Imaging-Massenspektrometrie zur Co-Lokalisation verschiedener Proteine mit Amyloid Ablagerungen in Nierenbiopsien

Aim: The main aim of this study is to identify amyloid proteins and establish their distribution in amyloidosis tissue sections using imaging mass spectrometry (IMS) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. A secondary aim was to investigate protein signals associated with amyloidosis disease. Methods: Renal FFPE biopsy specimens with AA-amyloidosis were cut (~10µm) and mounted onto regular slide for Congo red staining. Serial sections were cut (~2µm) and mounted onto ITO-coated conductive slides for IMS analysis. After deparaffination and antigen retrieval procedures, sections were on-tissue trypsin digested, sprayed with matrix using the ImagePrep device (Bruker Daltonik, Bremen), and subsequently analyzed by MALDI MS using a Bruker Autoflex Speed mass spectrometer (Bruker Daltonik, Bremen). Mass spectra data were loaded to ClinProTools software allowing profiles and statistical analysis to be directly linked to the histological image and subsequently compared to the IMS images for correlation study. Consecutive tissue sections were processed in parallel to provide both protein distribution and identification simultaneously. Peptides were extracted from tissues with 20 µL of buffer solution (10 % ACN, 1.1 % TFA) and subjected to LC-MS/MS analysis for protein identification. Results: Three peptides for serum amyloid A (SAA) (1456.7, 1612.8, 1670.7), and four peptides for serum amyloid P-component (SAMP) proteins (1156.6, 1166.6, 1406.7, and 1811.8) were identified that matched the masses assigned by IMS analysis. MS images were compared with optical images of the Congo-red serial tissue section resembling the amyloid deposition. Two proteins, vitronectin and apolipoprotein E, were identified and found to correlate with amyloid regions. Conclusion: IMS with LC-MS/MS analysis using on-tissue tryptic digestion allowed the identification of amyloidosis specific peptides and the precursor proteins providing information on both protein distribution and identification simultaneously. The association of vitronectin and apolipoprotein-E proteins with renal amyloid shown in this study suggests a role for these proteins in the process of amyloidogenesis. Previous studies demonstrated the contribution of vitronectin and apoliporpotein E proteins with the formation of amyloid protein1,2. 1. Shin TM et al. Mol Neurodegener. 2008 Oct 21;3:16 2. Kim J. et al. Exp Med. 2012 Nov 19;209(12):2149-56)