Laser capture microdissection in combination with mass spectrometry: Approach to characterization of tissue-specific proteomes of Eudiplozoon nipponicum (Monogenea, Polyopisthocotylea)

Eudiplozoon nipponicum (Goto, 1891) is a hematophagous monogenean ectoparasite which inhabits the gills of the common carp (Cyprinus carpio). Heavy infestation can lead to anemia and in conjunction with secondary bacterial infections cause poor health and eventual death of the host. This study is based on an innovative approach to protein localization which has never been used in parasitology before. Using laser capture microdissection, we dissected particular areas of the parasite body without contaminating the samples by surrounding tissue and in combination with analysis by mass spectrometry obtained tissue-specific proteomes of tegument, intestine, and parenchyma of our model organism, E. nipponicum. We successfully verified the presence of certain functional proteins (e.g. cathepsin L) in tissues where their presence was expected (intestine) and confirmed that there were no traces of these proteins in other tissues (tegument and parenchyma). Additionally, we identified a total of 2,059 proteins, including 72 peptidases and 33 peptidase inhibitors. As expected, the greatest variety was found in the intestine and the lowest variety in the parenchyma. Our results are significant on two levels. Firstly, we demonstrated that one can localize all proteins in one analysis and without using laboratory animals (antibodies for immunolocalization of single proteins). Secondly, this study offers the first complex proteomic data on not only the E. nipponicum but within the whole class of Monogenea, which was from this point of view until recently neglected.

This demonstrates why we opted for a different approach: MSI is well-suited to the 82 investigation of small molecules but lacks the ability to identify proteins whose size exceeds 83 app. 15 or 25 kDa [27,28], depending on the specific instrumental setup. 84 The ability to capture higher molecular weights is essential in search for functional 85 proteins, because their weight usually ranges around several tens of kilodaltons. For example, 86 the digestive peptidases of E. nipponicum, such as like cathepsins L and B, have molecular 87 weight of ~25 kDa, ~29 kDa, respectively [29], while one of the serine peptidase inhibitors 88 from the same organism has molecular weight of ~45 kDa [30]. 89 In the present study, our aim is first of all to investigate the potential of using laser 90 capture microdissection (LCM) and liquid chromatography coupled to tandem mass 91 spectrometry (LC-MS/MS) for protein localization and secondly, to obtain tissue-specific    To assess whether our approach could also be applied to protein localization, we 211 compared proteins identified in each tissue with previously characterized proteins whose

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Laser capture microdissection of tissue samples 218 We targeted 3 types of tissue -intestine, tegument and parenchyma (Fig 1). These protein content in intestine. In total, i.e. in all three tissues jointly, we identified 2,059 proteins.

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Of these proteins, 1,978 were found in the intestine, 1,425 in the tegument, and 1,302 in the 233 parenchyma. An overview is listed in Unique proteins from each tissue were assigned to KEGG pathways (Fig 3). Most  With respect to peptidases, we were able to identify 72 in all three tissues jointly.

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Numbers of peptidases identified in each tissue are shown in the Fig. 4. Most peptidases were 252 found in the intestine, where one also finds most of the unique ones. Unique inhibitors were sorted in several families according to MEROPS (Fig 7).    EnSerp1, were present in all tissues (see Table 3).

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In this study, we report the first tissue-specific proteomic analysis of parasitic 305 monogenean Eudiplozoon nipponicum. Using laser capture microdissection, we dissected 306 particular areas of three different tissues of the parasite body and the dissected samples were 307 subjected to mass spectrometry-based proteomic analysis. This allowed us to characterize 308 tissue-specific proteomes and to assess the suitability of this approach for proteins tissue-   Fig 2). Of these proteins, 72 (3.50%) 319 were peptidases and 33 (1.60%) inhibitors (Table 1)

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In terms of tissue specificity of peptidases and inhibitors (Fig 4 and 6), the richest further support to these conclusions (Fig 3) The catalytic groups, which cover the unique identified peptidases, are shown in Fig 5.

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In total, we found representatives of four groups. Most represented were serine, cysteine, and 346 metallopeptidases. This correlates with the number of unique inhibitors, which were assigned 347 to seven families, including representatives of metallo (I87), cysteine (I29, I25, I4), and serine 348 (I4 and I2) peptidase inhibitors (Fig 7). In all these statistics, the intestine was once again 349 dominant with respect to variety. This is once again related to its function, namely feeding,

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This is reflected in case of the most abundant peptidases/inhibitors (Table 2). For 353 example, in the intestine we found serine carboxypeptidase. Serine carboxypeptidases from 354 family S10 are known to have a lysosomal function [58], but it is also assumed that they have 355 a role in blood degradation. The latter function seems to be supported by findings on S. In the parenchyma, one functional protein is significantly abundant: an inhibitor from 366 the I63 family. This family contains an inhibitor of metallopeptidase pappalysin-1, which 367 promotes cell growth [63]. We can therefore speculate that the identified inhibitor may play a 368 role in the regulation of a peptidase with similar function.

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Moreover, since we have access to the preliminary version of secretome of 370 E. nipponicum (manuscript in preparation), we compared our data with it and saw that most 371 secretome proteins are in fact a subset of data obtained in the present study. This is unsurprising 372 because proteins in the secretome are produced by the parasite's glands and intestine and at the 373 time of production, they are situated in the parasite body, which is why they were detected by 374 mass spectrometry in our analysis.

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Finally, one of the main goals of this study was to investigate the potential of application  Table   386 3). On the other hand, there is still room for improvement because two of the eleven examined 387 cathepsins were not identified and another two were identified in tissues where they were not 388 expected.