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The aim is to improve in situ tryptic digestion for MALDI\u2013MSI of FFPE samples and determine if similar results would be reproducible if obtained from different sites.<\/jats:p><\/jats:sec><jats:sec><jats:title>Experimental Design<\/jats:title><jats:p>FFPE tissues (mouse intestine, human ovarian teratoma, tissue microarray of tumor entities sampled from three different sites) are prepared for MALDI\u2013MSI. Samples are coated with trypsin using an automated sprayer then incubated using deliquescence to maintain a stable humid environment. After digestion, samples are sprayed with CHCA using the same spraying device and analyzed with a rapifleX MALDI Tissuetyper at 50\u00a0\u00b5m spatial resolution. Data are analyzed using flexImaging, SCiLS, and R.<\/jats:p><\/jats:sec><jats:sec><jats:title>Results<\/jats:title><jats:p>Trypsin application and digestion are identified as sources of variation and loss of spatial resolution in the MALDI\u2013MSI of FFPE samples. Using the described workflow, it is possible to discriminate discrete histological features in different tissues and enabled different sites to generate images of similar quality when assessed by spatial segmentation and PCA.<\/jats:p><\/jats:sec><jats:sec><jats:title>Conclusions and Clinical Relevance<\/jats:title><jats:p>Spatial resolution and site\u2010to\u2010site reproducibility can be maintained by adhering to a standardized MALDI\u2013MSI workflow.<\/jats:p><\/jats:sec>","DOI":"10.1002\/prca.201800029","type":"journal-article","created":{"date-parts":[[2018,11,8]],"date-time":"2018-11-08T18:32:36Z","timestamp":1541701956000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":83,"title":["Site\u2010to\u2010Site Reproducibility and Spatial Resolution in MALDI\u2013MSI of Peptides from Formalin\u2010Fixed Paraffin\u2010Embedded Samples"],"prefix":"10.1002","volume":"13","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-0827-6840","authenticated-orcid":false,"given":"Alice","family":"Ly","sequence":"first","affiliation":[{"name":"Bruker Daltonik GmbH  Bremen Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"R\u00e9mi","family":"Longuesp\u00e9e","sequence":"additional","affiliation":[{"name":"Institute of Pathology University Hospital Heidelberg  Heidelberg Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9054-4786","authenticated-orcid":false,"given":"Rita","family":"Casadonte","sequence":"additional","affiliation":[{"name":"Proteopath GmbH  Trier Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Petra","family":"Wandernoth","sequence":"additional","affiliation":[{"name":"Proteopath GmbH  Trier Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3717-0823","authenticated-orcid":false,"given":"Kristina","family":"Schwamborn","sequence":"additional","affiliation":[{"name":"Institute of Pathology Technical University of Munich  Munich Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Christine","family":"Bollwein","sequence":"additional","affiliation":[{"name":"Institute of Pathology Technical University of Munich  Munich Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Christian","family":"Marsching","sequence":"additional","affiliation":[{"name":"Center for Biomedical Mass Spectrometry and Optical Spectroscopy (CeMOS) Mannheim University of Applied Sciences  Mannheim Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Katharina","family":"Kriegsmann","sequence":"additional","affiliation":[{"name":"Department of Hematology Oncology and Rheumatology University Hospital Heidelberg  Heidelberg Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Carsten","family":"Hopf","sequence":"additional","affiliation":[{"name":"Center for Biomedical Mass Spectrometry and Optical Spectroscopy (CeMOS) Mannheim University of Applied Sciences  Mannheim Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wilko","family":"Weichert","sequence":"additional","affiliation":[{"name":"Institute of Pathology Technical University of Munich  Munich Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"J\u00f6rg","family":"Kriegsmann","sequence":"additional","affiliation":[{"name":"Proteopath GmbH  Trier Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Peter","family":"Schirmacher","sequence":"additional","affiliation":[{"name":"Institute of Pathology University Hospital Heidelberg  Heidelberg Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7319-3646","authenticated-orcid":false,"given":"Mark","family":"Kriegsmann","sequence":"additional","affiliation":[{"name":"Institute of Pathology University Hospital Heidelberg  Heidelberg Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"S\u00f6ren\u2010Oliver","family":"Deininger","sequence":"additional","affiliation":[{"name":"Bruker Daltonik GmbH  Bremen Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2019,1,4]]},"reference":[{"key":"e_1_2_8_1_1","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0098187"},{"key":"e_1_2_8_2_1","doi-asserted-by":"publisher","DOI":"10.1038\/ki.2012.108"},{"key":"e_1_2_8_3_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.bbapap.2016.10.006"},{"key":"e_1_2_8_4_1","author":"Smith A.","year":"2018","journal-title":"J. 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