Investigation of neutrophilic proteins/peptides in periprosthetic tissue by matrix-assisted laser desorption/ionization (MALDI) imaging

Background: The diagnosis of infections in patients with arthritis and/or in joint prostheses requires interdisciplinary cooperation and the application of up-to-date methods. Histological evaluation of the synovial membrane is made by counting neutrophils per area of tissue, giving a highly imprecise and unsatisfactory diagnosis. Because of the low sensitivity of histochemical methods, molecular approaches increases the diagnostic accuracy in the differential diagnosis of granulomatous synovitis, especially in mycobacterial infections, allowing the detection of bacterial DNA or RNA in synovial fluid or tissues [1]. Proteomic methods could increment the methodological spectrum for the diagnosis of infections. Aim of this study is to establish a MALDI imaging method that can be applied to the routine investigation for screening prosthesis lesions. Methods: Periprosthetic FFPE tissue samples containing a very high number of neutrophils were on-tissue digested with trypsin (0.1µg/µL), and sprayed with a matrix solution for MALDI analysis. MALDI spectra were exported from regions with high number of neutrophils and statistically compared with spectra collected from areas containing a low number of neutrophils using ClinProTools (Bruker Daltonik) and SCiLS Lab (SCiLS GmbH) software. Results: Several signal peptides with high intensity were mainly detected in tissue regions with a high amount of neutrophils. Among those signals, m/z 788.405 was detected as predictive peptides for calgranulin C (S100-A12), m/z 660.273 for annexin-A1, m/z 944.533 for histone H2A, and m/z 1349.621 for calgizzarin (S100-A11). The co-localized masses resembled the area with high number of neutrophils showed in the histochemical image. Conclusion: We previously detected neutrophil enzymes including granzymes and members of the S100-protein group on fresh-frozen inflamed synovial tissues [2]. Here we have applied MALDI IMS on FFPE tissues that generated a proteomic pattern distinguishing patients with high-grade from low-grade synovitis. Furthermore, identification of the peptides is urgently required to provide a reliable diagnostic test. References: 1.J. Kriegsmann et al., [Molecular pathological diagnostics of infections in orthopedic pathology]. Pathologe. 2014 Nov;35 Suppl 2:225-31. 2.M. Kriegsmann et al., [MALDI MS imaging as a powerful tool for investigating synovial tissue]. Scand J Rheumatol. 2012 Aug;41(4):305-9.