MALDI imaging mass spectrometry (MALDI-IMS): a new approach for spatial identification of proteins in feline oviducts

The knowledge and understanding of many reproductive processes have improved significantly over the years, especially after the development of tools for protein identification1. The traditional approach for spatial identification of proteins requires a prior knowledge of the target entities and narrows the number of targets that can be monitored simultaneously1. For that reason, MALDI imaging mass spectrometry (MALDI-IMS) once it is able to directly scan the target tissue, is considered an outstanding approach for molecular profiling and imaging. With the purpose to identify the factors involved in early stages of embryo development in the domestic cat, this technology was used for the first time to analyse the spatial distribution of proteins in oviducts of queens. Reproductive tracts were collected from 2 queens (cross-breed, 2 and 4 years old) undergoing routine ovariohysterectomy. The oviducts were carefully dissected, divided into three segments (distal to the ovary, proximal to the ovary and the mid-section between the two), snap-frozen in liquid nitrogen and stored at -80ºC until use. Then, they were sectioned (11 μm) in a cryostat and fixed on ITO (indium tin oxide) conductive glass slides while serial sections were collected on microscope slides for haematoxylin and eosin staining. For MALDI-IMS, samples were coated with a thin homogeneous layer of CHCA (α-Cyano-4-hydroxycinnamic acid) matrix using a nebulization device. MALDI images were acquired on an Autoflex III Smartbeam instrument (Bruker Daltonics) with 400 shots/spectrum, over the mass range of m/z 2 to 20 kDa in the positive ion mode. The spatial resolution of images was 80 μm. The molecular images obtained in this study show that protein distribution was different in the oviductal infundibulum, ampulla, and isthmus identified by histology. Mass spectra was characterized by abundant ions of m/z 1259, 4939, 4960 and 10626. Based on the literature2 these ions might correspond to keratin, thymosin β10, thymosin β4 and S100, respectively. S100 proteins are calcium modulated proteins implicated in a variety of cellular activities, including cell differentiation and regulation of cell motility. Keratin and thymosins are involved in the biological response to tissue damage. In women, this defence system is adapted to operate in the genital tract, being altered according to the hormonal and cellular changes that occur during menstruation (characterized by cell death and necrosis)3. Protein attributions described herein must be confirmed by MS/MS experiments, but our results indicate that it is possible to use this technology to elucidate key molecular processes involved in the reproductive physiology of carnivore species. [1] Lagarreigue M, Lavigne R, Guevel B, et al. Matrix-Assisted Laser Desorption/Ionization Imaging Mass Spectrometry: A Promising Technique for Reproductive Research. Biology of Reproduction 2012; 86(3): 1–11. [2] McDonnell LA, Walch A. Stoeckli M, Corthals GL. MSiMass list: a public database of identifications for protein MALDI MS imaging. Journal of proteome research 2013, 13 (2), 1138-1142. [3] Horne AW, Stock SJ, King AE. Innate immunity and disorders of the female reproductive tract. Reproduction 2008; 135(6): 739-749.