{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,1,23]],"date-time":"2026-01-23T20:21:57Z","timestamp":1769199717020,"version":"3.49.0"},"reference-count":21,"publisher":"MDPI AG","issue":"9","license":[{"start":{"date-parts":[[2021,8,27]],"date-time":"2021-08-27T00:00:00Z","timestamp":1630022400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"ZIM","award":["ZF4380802"],"award-info":[{"award-number":["ZF4380802"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Pharmaceutics"],"abstract":"<jats:p>Sepsis is the number one cause of death in intensive care units. This life-threatening condition is caused by bacterial infections and triggered by endotoxins of Gram-negative bacteria that leads to an overreaction of the immune system. The synthetic anti-lipopolysaccharide peptide Pep19-2.5 is a promising candidate for the treatment of sepsis as it binds sepsis-inducing lipopolysaccharides and thus prevents initiation of septic shock. For clinical evaluation precise quantification of the peptide in blood and tissue is required. As the peptide is not extractable from biological samples by commonly used methods there is a need for a new analysis method that does not rely on extraction of the peptide. In order to quantify the peptide by mass spectrometry, the peptide was synthesized containing 13C9,15N1-labeled phenylalanine residues. This modification offers high stability during acidic hydrolysis. Following acidic hydrolysis of the samples, the concentration of 13C9,15N1-labeled phenylalanine determined by LC-MS could be unambiguously correlated to the content of Pep19-2.5. Further experiments validated the accuracy of the data. Moreover, the quantification of Pep19-2.5 in different tissues (as studied in Wistar rats) was shown to provide comparable results to the results obtained with radioactively-labeled (14C) Pep19-2.5- Radioactive labeling is considered as the gold standard for quantification of compounds that refrain from reliable extraction methods. This novel method represents a valuable procedure for the determination of Pep19-2.5 and sticky peptides with unpredictable extraction properties in general.<\/jats:p>","DOI":"10.3390\/pharmaceutics13091342","type":"journal-article","created":{"date-parts":[[2021,8,31]],"date-time":"2021-08-31T04:07:28Z","timestamp":1630382848000},"page":"1342","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Mass Spectrometric Quantification of the Antimicrobial Peptide Pep19-2.5 with Stable Isotope Labeling and Acidic Hydrolysis"],"prefix":"10.3390","volume":"13","author":[{"given":"Sabrina","family":"Wohlfart","sequence":"first","affiliation":[{"name":"Department of Nuclear Medicine, Heidelberg University Hospital, 69120 Heidelberg, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Michael","family":"Kilian","sequence":"additional","affiliation":[{"name":"Department of Nuclear Medicine, Heidelberg University Hospital, 69120 Heidelberg, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Philip","family":"Storck","sequence":"additional","affiliation":[{"name":"Department of Nuclear Medicine, Heidelberg University Hospital, 69120 Heidelberg, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Thomas","family":"Gutsmann","sequence":"additional","affiliation":[{"name":"Division of Biophysics Research Center Borstel, Leibniz-Center for Medicine and Bioscience, 23845 Borstel, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Klaus","family":"Brandenburg","sequence":"additional","affiliation":[{"name":"Brandenburg Antiinfektiva GmbH, Parkallee 10b, 23845 Borstel, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Walter","family":"Mier","sequence":"additional","affiliation":[{"name":"Department of Nuclear Medicine, Heidelberg University Hospital, 69120 Heidelberg, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2021,8,27]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"119","DOI":"10.1146\/annurev.biochem.78.082907.145923","article-title":"Multidrug resistance in bacteria","volume":"78","author":"Nikaido","year":"2009","journal-title":"Annu. Rev. Biochem."},{"key":"ref_2","first-page":"19688","article-title":"Methicillin-Resistant Staphylococcus Aureus (MRSA): Burden of Disease and Control Challenges in Europe","volume":"15","author":"Kock","year":"2010","journal-title":"Euro Surveill. Bull. Eur. Sur Les Mal. Transm. Eur. Commun. Dis. Bull."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"572","DOI":"10.1136\/postgradmedj-2015-133475","article-title":"Puerperal sepsis in the 21st century: Progress, new challenges and the situation worldwide","volume":"91","author":"Buddeberg","year":"2015","journal-title":"Postgrad. Med. J."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"427","DOI":"10.1016\/j.mib.2003.08.003","article-title":"Why is big Pharma getting out of antibacterial drug discovery?","volume":"6","author":"Projan","year":"2003","journal-title":"Curr. Opin. Microbiol."},{"key":"ref_5","first-page":"18","article-title":"Antibacterial drug discovery: Is it all downhill from here?","volume":"10","author":"Projan","year":"2004","journal-title":"Clin. Microbiol. Infect. Off. Publ. Eur. Soc. Clin. Microbiol. Infect. Dis."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"55","DOI":"10.1007\/s10353-014-0253-0","article-title":"Antibiotic resistance: A long term, serious problem\u2026getting worse. Thoughts on the future of surgery in a post-antibiotic era","volume":"46","author":"Loughran","year":"2014","journal-title":"Eur. Surg."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"887","DOI":"10.1038\/jid.2011.387","article-title":"Antimicrobial Peptides: Old Molecules with New Ideas","volume":"132","author":"Nakatsuji","year":"2012","journal-title":"J. Investig. Dermatol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"379","DOI":"10.1128\/CMR.16.3.379-414.2003","article-title":"Receptors, mediators, and mechanisms involved in bacterial sepsis and septic shock","volume":"16","author":"Kuiper","year":"2003","journal-title":"Clin. Microbiol. Rev."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"840","DOI":"10.1056\/NEJMra1208623","article-title":"Severe Sepsis and Septic Shock","volume":"369","author":"Angus","year":"2013","journal-title":"N. Engl. J. Med."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"3817","DOI":"10.1128\/AAC.00534-10","article-title":"New Antiseptic Peptides To Protect against Endotoxin-Mediated Shock","volume":"54","author":"Gutsmann","year":"2010","journal-title":"Antimicrob. Agents Chemother."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"R3","DOI":"10.1186\/cc11920","article-title":"The anti-inflammatory effect of the synthetic antimicrobial peptide 19-2.5 in a murine sepsis model: A prospective randomized study","volume":"17","author":"Schuerholz","year":"2013","journal-title":"Crit. Care"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"14292","DOI":"10.1038\/srep14292","article-title":"Lipoproteins\/peptides are sepsis-inducing toxins from bacteria that can be neutralized by synthetic anti-endotoxin peptides","volume":"5","author":"Heinbockel","year":"2015","journal-title":"Sci. Rep."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"1576","DOI":"10.1111\/febs.14805","article-title":"Antibacterial action of synthetic antilipopolysaccharide peptides (SALP) involves neutralization of both membrane-bound and free toxins","volume":"286","author":"Correa","year":"2019","journal-title":"FEBS J."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"1480","DOI":"10.1128\/AAC.02066-12","article-title":"Preclinical Investigations Reveal the Broad-Spectrum Neutralizing Activity of Peptide Pep19-2.5 on Bacterial Pathogenicity Factors","volume":"57","author":"Heinbockel","year":"2013","journal-title":"Antimicrob. Agents Chemother."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"689","DOI":"10.1016\/j.jsps.2015.01.023","article-title":"Comparison of different serum sample extraction methods and their suitability for mass spectrometry analysis","volume":"23","author":"Alshammari","year":"2015","journal-title":"Saudi Pharm. 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