Aim: Rheumatoid arthritis (RA) is an autoimmune disease with proliferation of lymphocytes, fibroblasts and synovial lining, causing cartilage and bone destruction. The etiology and pathogenesis of this disease are still unknown. Early and precise diagnosis is mandatory to choose the appropriate therapy. Diagnosis of RA is made according to the ACR-criteria (American Congress of Rheumatology). Personalized medicine requires exact description of synovial pathology to target the appropriate cell population in various RA subtypes or stages. The aim of this experimental study is to identify differences in peptide patterns in two compartments, lining and sublining, of synovial tissue by MALDI Imaging Mass Spectrometry (MS). Methods: Patients were classified according to the ACR criteria for RA. One section of a tissue sample was collected onto a MALDI target slide and an sdjacent serial FFPE section was stained with H&E andreviewed by a pathologist. Areas of interest (synovial lining and sublining layer, 10 discrete areas per compartment) were digitally marked, and the histology annotated images were merged with a photomicrograph of the section taken for the MALDI MS measurement. For MALDI MS analysis, trypsin and matrix solution were applied to discrete regions of interest using a robotic spotter. Pixel coordinates of these areas were transferred to a mass spectrometer for spectral acquisition in reflectron mode. Mass spectra from marked areas were exported and loaded into ClinProTools 3.0 software (Bruker Daltonik GmbH) for statistical analysis. Pairwise statistical comparison was performed on the basis of P-values from the Wilcoxon/Kruskal-Wallis test (p<0.05) and with a 99.5% of confidence level and at 1.7 fold change. 20 tissue sections were analyzed. Some tissue sections were chosen for MALDI Imaging MS analysis to show the distribution of the m/z values of interest. Results: Tryptic peptide molecules, visualized as peaks in the mass spectra with specific mass-to-charge (m/z) values, were measured in the two tissue compartments. Preliminary data showed strong differences in the relative intensity of ions obtained in the synovial lining layer (m/z 537.3, 650.1, 656.1, 666.1, 672.1, 704.4, 710.4, 726.5, 855.2, 856.2, 861.2, 864.6, 877.1, 916.0, 932.1, 1066.2). Conclusion: MALDI Imaging MS revealed 16 different molecular signatures discriminating synovial lining from sublining layer. Further studies are required to provide greater statistical power and to identify subtypes or different disease stages that are directly applicable to personalized medicine.
MALDI Imaging Mass Spectrometry to Investigate Compartments of Synovial Tissue of Patients with Rheumatoid Arthritis
Casadonte R;
2013-01-01
Abstract
Aim: Rheumatoid arthritis (RA) is an autoimmune disease with proliferation of lymphocytes, fibroblasts and synovial lining, causing cartilage and bone destruction. The etiology and pathogenesis of this disease are still unknown. Early and precise diagnosis is mandatory to choose the appropriate therapy. Diagnosis of RA is made according to the ACR-criteria (American Congress of Rheumatology). Personalized medicine requires exact description of synovial pathology to target the appropriate cell population in various RA subtypes or stages. The aim of this experimental study is to identify differences in peptide patterns in two compartments, lining and sublining, of synovial tissue by MALDI Imaging Mass Spectrometry (MS). Methods: Patients were classified according to the ACR criteria for RA. One section of a tissue sample was collected onto a MALDI target slide and an sdjacent serial FFPE section was stained with H&E andreviewed by a pathologist. Areas of interest (synovial lining and sublining layer, 10 discrete areas per compartment) were digitally marked, and the histology annotated images were merged with a photomicrograph of the section taken for the MALDI MS measurement. For MALDI MS analysis, trypsin and matrix solution were applied to discrete regions of interest using a robotic spotter. Pixel coordinates of these areas were transferred to a mass spectrometer for spectral acquisition in reflectron mode. Mass spectra from marked areas were exported and loaded into ClinProTools 3.0 software (Bruker Daltonik GmbH) for statistical analysis. Pairwise statistical comparison was performed on the basis of P-values from the Wilcoxon/Kruskal-Wallis test (p<0.05) and with a 99.5% of confidence level and at 1.7 fold change. 20 tissue sections were analyzed. Some tissue sections were chosen for MALDI Imaging MS analysis to show the distribution of the m/z values of interest. Results: Tryptic peptide molecules, visualized as peaks in the mass spectra with specific mass-to-charge (m/z) values, were measured in the two tissue compartments. Preliminary data showed strong differences in the relative intensity of ions obtained in the synovial lining layer (m/z 537.3, 650.1, 656.1, 666.1, 672.1, 704.4, 710.4, 726.5, 855.2, 856.2, 861.2, 864.6, 877.1, 916.0, 932.1, 1066.2). Conclusion: MALDI Imaging MS revealed 16 different molecular signatures discriminating synovial lining from sublining layer. Further studies are required to provide greater statistical power and to identify subtypes or different disease stages that are directly applicable to personalized medicine.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
