Microsoft Word - ISJ430   ISJ 13: 205-209, 2016 ISSN 1824-307X SHORT COMMUNICATION Application of MALDI-MSI for detection of antimicrobial peptides in tissues of the marine invertebrate Arenicola marina AL Maltseva1, VV Starunov1, PA Zykin2 1Department of Invertebrate Zoology, St Petersburg State University, St Petersburg, Russia 2Department of Cytology and Histology, St Petersburg State University, St Petersburg, Russia Accepted June 13, 2016 Abstract MALDI imaging mass-spectrometry (MALDI-MSI) is a highly informative approach combining morphology with molecular data. It is widely applied in neuroscience, plant science, cancer-biology, biomedicine, including clinical, and preclinical studies, but not for investigation of endogenous peptides/proteins or metabolites in marine invertebrates. We examined the informativeness of MALDI- MSI for analysis of distribution of antimicrobial peptides (arenicins) in the polychete Arenicola marina and concluded that it can be successfully used as a primary rough express screening method. Key Words: MALDI-MSI; antimicrobial peptides; polychete immunity; Arenicola marina   Introduction Antimicrobial peptides (AMPs) are key players in innate immunity of diverse organisms, contributing to both effector and regulatory functions. They are among immune effectors most intensively studied during last 30 years (Harder and Schröder, 2016). AMPs are relatively small (not exceeding 100 amino acids) usually cationic polypeptidic molecules with a prominent inhibitory potential against various microbial pathogens. Although AMPs were identified in a wide range of organisms including plants, vertebrate and invertebrate animals, protists and prokaryotes (Boman, 2003; Reddy et al., 2004; Yount et al., 2006; Otero-González et al., 2010; Pasupuleti et al., 2012; Harder and Schröder, 2016) different taxa are still very unequally studied in terms of AMPs diversity and functioning, and immunology in general. The wide distribution of AMPs makes them an attractive object for comparative immunology and description of AMPs functioning in less studied taxa (among which many marine invertebrates, e.g., polychetes) is in high demand. Being very important in respect of both practical and theoretical points of view, AMPs attract attention in different aspects: structure, structure- functional interrelation, mechanism of action, spatiotemporal pattern of expression and mechanism ___________________________________________________________________________ Corresponding author: Arina L Maltseva Department of Invertebrate Zoology St Petersburg State University Universitetskaya 7/9, St Petersburg, 199034, Russia E-mail: arina.maltseva@spbu.ru of its regulation. In this respect, the widening of methodological background for AMPs investigation in different taxa is an urgent task. MALDI imaging mass-spectrometry (MALDI- MSI) is a comparatively young and highly informative approach superposing morphological and molecular data, which was called "molecular histology" (Stoeckli et al., 2001; Walch et al., 2008). It allows characterization of spatial distribution of a wide spectrum of components (such as proteins, peptides, lipids, glycanes, hormones, secondary metabolites) in situ in a crude tissue material label free, without laborious procedures of processing histochemical slides, obtaining antibody and staining. The pioneering applications of MALDI-MSI for analysis of specific peptides in whole cells were performed on neuronal tissue of snails (Jimenez et al., 1994; Dreisewerd et al., 1997). Nearly in that period, the effectiveness of MALDI-MSI usage in fresh tissue sections was proved (Caprioli et al., 1997). Since then MALDI-MSI became a routine method in neuroscience, plant science, cancer- biology, biomedicine, including clinical and preclinical studies (e.g., Baluff et al., 2011; Salzet et al., 2012). Recent advances include in situ trypsinization for protein profiling, even in formalin- fixed tissues (De Sio et al., 2015), the use of specialized matrices for small molecules (Shanta et al., 2012) and in situ derivation for direct detection of some neuromediators (Shariatgorji et al., 2015). However, the applications of MALDI-MSI for investigation of endogenous peptides/proteins or metabolites in invertebrates are still not so numerous (e.g., Esquenazi et al., 2008; Bruand et 205    mailto:arina.maltseva@spbu.ru   Fig. 1 MALDI-MSI visualization of arenicins (2,758.3 Da ± 2Da) in anterior (A, B) and main body (C, D, E) parts. A, C - densitometry imaged slices; B, D - arenicin-positive MS-signal in anterior or main body parts, respectively; E - representation of principal body compartments by filtered MS-signals: celomic fluid (arenicins 2,758.3 Da ± 2Da, red channel), epithelium of body wall (m/z 1,212.9 ± 1, blue channel) and celothelium (1,006.3 ± 1, green channel). al., 2011); in respect to AMPs of invertebrates they are unique (Kuhn-Nentwig et al., 2014). This motivated us to examine the applicability and informative value of MALDI-MSI for characterization of distribution of AMPs (arenicins) in the marine invertebrate Arenicola marina. Material and Methods Animals Adult lugworms (approx. 15 cm length) Arenicola marina were collected from wild populations in the intertidal zone in the vicinity of the White Sea Marine biological station of Saint Petersburg University (Russia). Animals were maintained in permanently aerating static tanks with marine water for 5 - 7 days. Tissue preparation For preparation of cryo-slides worms were frozen in liquid nitrogen after being anesthetized in 5 % MgCl2 in sterile marine water. Immediately after freezing, cross-sections (15 µm thick) of anterior (first chaetigerous segments) and middle body (gill- carrying segments) parts were prepared with Cryomicrotome (Leica CM 3050S) at -18 °C. Sections were thaw mounted onto indium tin oxide coated glass slides and drayed under vacuum in Labconco SpeedVac for 30 min. Fiducial points for microscopic and MALDI-MSI co-registration were drawn with Edding 750 white marker and preparations were subsequently imaged with Bio- Rad GS-800 calibrated densitometer at 600 dpi. For calibration purposes, Bruker Peptide Calibration Standard II was deposited near feducual points. Sections were matrix-coated in automated matrix sprayer Bruker ImagePrep (program version 2.0.1) using build-in “HCCA_nsh04” protocol with matrix solution containing 7 g/l a-Cyano-4-hydroxy- cinnamic acid (CAS 28166-41-8), 50 % v/v acetonitrile and 0.2 % v/v trifluoroacetic acid. MALDI-pictures obtaining and processing. Spectra were obtained on MALDI-TOF Bruker Ultraflextreme mass-spectrometer with Bruker FlexControl v. 3.3 and FlexImaging v. 3.0 software. Laser intensity was set to 60 %, laser spot size was set to “small”, raster size 100 µm. Actual laser spot size measured by matrix ablation was around 10 µm. Spectra were collected in reflected mode with positive polarity, mass window 700 - 3,500 Da, pulsed ion extraction delay 80 ns, matrix ions with masses less than 700 Da were deflected. The preparation was scanned in bidirectional mode with 1000 shoots per pixel and random walk pattern inside pixel. MALDI-MSI and optical images were aligned in FlexImaging by previously marked fiducial points. Method was calibrated on spots previously 206      Fig. 2 Hematoxylin-eosin staining after MALDI-imaging. A, B - cross-sections through anterior (A) and middle (B) parts of the body; C - fragment of the body wall; D - longitudinal body musculature and celothelium; E, F - celomocytes. Abbreviations: c - celomic cavity, cc - celomocytes, ch - chaetae, cm - circular body musculature, coe - celomic epithelium, cu - cuticle, cue - cuticular epithelium, d - dissepiment, dbv - dorsal blood vessel, g - gut, lm - longitudinal body musculature, vbv - ventral blood vessel, vnc - ventral nerve cord. deposited with Bruker Peptide Calibration Standard II. Spectra were preprocessed by baseline subtraction using TopHat algorithm, no smoothing was done, spectra were normalized by total ion count (TIC), peak-picking was done with SNAP algorithm. Histology and light microscopy After MALDI imaging slides were washed in ethanol to remove the matrix, stained with Ehrlich’s hematoxylin-eosin following standard protocol and mounted in DPX mounting medium. The slides were examined with Leica DM2500 light microscope equipped by Leica DFC495 camera. Results and Discussion Arenicins are short (21 residue peptides, 2.8 kDa) peptides with strong antimicrobial activity, originally isolated from motile phagocytic cells of celomic fluid - celomocytes (Ovchinnikova et al., 2004). The spatiotemporal pattern of arenicins expression in lugworm body was later thoroughly characterized with immunohistochemistry and semi- quantitative RT-PCR. Although the expression of arenicins takes place in different compartments, the strongest signal was detected in cells of celomic fluid (Maltseva et al., 2014). This allows to compare present results with earlier work to determine the applicability and scope of the approach chosen here. MALDI-MSI is used in either linear or reflected mode, the later with higher resolution. Arenicins are short enough to be analyzed in reflected mode of TOF/MS with resolution up to 20,000. Identification of arenicins was done by previously known by LC- ESI-qTOF mass of 2,758.3 Da with mass window ± 2Da (Figs 1B, D, E - red channel). For better structure representation automatic mass list filtering was done revealing two m/z of 1,212.9 ±1 and 1,006.3 ±1 colocalized with epithelium of body wall and celothelium respectively (Fig.1E - blue and green channels). Correspondence of MS-signals to particular tissues/cells was established on the basis of histological staining of MALDI-scanned slides (Fig. 2). The staining indicates acceptable preservation of all compartments from which reliable signals were detected - epithelium and musculature of the body wall (2C), celothelium (2D) and its derivates, gut (2A, B), celomocytes (2E, F). Figure 1 shows that arenicin-positive signal in both anterior (1a, b) and main body (1C, D, E) parts was detected in celomic fluid, where strong arenicins expression was described. No specific signal was obtained from other body compartments, where arenicins were reported to be expressed (epithelium of the body wall and gut, extravasal 207      tissue, cuticle, ventral nerve cord (Maltseva et al., 2014). The result obtained with MALDI-MSI only partially reproduced the previously reported data. There could be several possible not mutually exclusive explanations for this. (1) Differential abundance of arenicins in different tissues. Previous results demonstrated that the arenicins are more abundant in celomocytes than in other cells types (Maltseva et al., 2014). The strength of the signal from body cavity was not maximal, but moderate (which could be the negative consequence of salt presence in tissues of marine animals). This allows to suspect that in the other compartments the abundance of arenicins was below the limit of detection. Similarly, only AMPs ctenidins highly abundant within granules of spider hemocytes were detectable by MALDI-MSI, but not defensins, stored in small amounts (Kuhn-Nentwig et al., 2014). (2) Differential accessibility of arenicins in different tissues. Celomocytes of polychetes are rather fragile cells. Thus, they are easily destroyed during contact with exogenous material as was reported, e.g., for celomocytes of Nereis diversicolour during the process of implant encapsulation (Porchet-Hennere et al., 1987). This is also the case for A. marina celomocytes (our own observation). So, possibly arenicins are more easily extracted into the matrix from celomocytes than from other tissues - this makes them detectable in these cells. This explanation does not exclude the first one. Conclusion The first application of MALDI-MSI for study AMPs in marine invertebrates was performed and reported here. The obtained data additionally confirm that celomocytes are the major compartment of arenicins expression. Although the present results only partially reproduced the output of alternative methods, the main compartment of arenicins expression was correctly detected. Consequently, MALDI-MSI can be successfully used as primary rough express screening approach for characterization of distribution of AMPs in tissues of marine invertebrates. Acknowledgments The opportunities for MALDI-MSI were provided by “Center for Molecular and Cell Technologies” Research Park, St. Petersburg State University, Russia. The work was supported by the Russian Foundation for Basic Research (RFBR, research grant № 16-34-60134). We thank Dr M Varfolomeeva for comments on the manuscript. References Balluff B, Schöne C, Höfler H, Walch A. 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