{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,4]],"date-time":"2026-08-04T12:48:26Z","timestamp":1785847706468,"version":"3.56.0"},"reference-count":27,"publisher":"American Chemical Society (ACS)","issue":"30","license":[{"start":{"date-parts":[[2026,7,21]],"date-time":"2026-07-21T00:00:00Z","timestamp":1784592000000},"content-version":"stm-asf","delay-in-days":0,"URL":"https:\/\/doi.org\/10.15223\/policy-029"},{"start":{"date-parts":[[2026,7,21]],"date-time":"2026-07-21T00:00:00Z","timestamp":1784592000000},"content-version":"stm-asf","delay-in-days":0,"URL":"https:\/\/doi.org\/10.15223\/policy-037"},{"start":{"date-parts":[[2026,7,21]],"date-time":"2026-07-21T00:00:00Z","timestamp":1784592000000},"content-version":"stm-asf","delay-in-days":0,"URL":"https:\/\/doi.org\/10.15223\/policy-045"}],"funder":[{"DOI":"10.13039\/501100004359","name":"Vetenskapsr?det","doi-asserted-by":"publisher","award":["2022-00841"],"award-info":[{"award-number":["2022-00841"]}],"id":[{"id":"10.13039\/501100004359","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100011077","name":"Sk?nes universitetssjukhus","doi-asserted-by":"publisher","id":[{"id":"10.13039\/501100011077","id-type":"DOI","asserted-by":"publisher"}]},{"name":"ALF-Agreement","award":["2022-0205"],"award-info":[{"award-number":["2022-0205"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2026,8,4]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>Quantitative LC\u2013ESI\u2013MS\/MS methods for peptides are conventionally developed and performed under acidic mobile-phase conditions to favor protonation and stable precursor formation. However, despite this long-standing practice, an increasing number of studies indicate that electrospray ionization (ESI) efficiency is often decoupled from bulk-solution protonation equilibria. Here, we systematically evaluate peptide-dependent ionization behavior under acidic (pH \u2248 2.8) and basic (pH \u2248 8.9) conditions using a dual-pH LC\u2013MS\/MS workflow applied to human coagulation factor VIII (FVIII). Fourteen FVIII signature peptides were generated by tryptic digestion and quantified using a fixed doubly charged precursor to ensure methodological consistency. Despite being predicted to be neutral or strongly anionic in bulk solution at pH 8.9, several FVIII peptides produced their highest signal under basic conditions, including peptides that were weakly detected or undetectable using acidic workflows. Comparison of the observed responses with estimated solution-phase charge shows that neither bulk protonation nor isoelectric point reliably predicts ESI performance. Instead, the data indicate that peptide response reflects peptide-intrinsic properties, notably hydrophobic surface activity and gas-phase proton affinity, which together govern access to the electrospray droplet interface and stabilization of charge during desolvation. Basic mobile phases selectively enhanced ESI+ response for peptides that interacted less favorably with the bulk aqueous phase, revealing a robust \u201cwrong-way-round\u201d ionization regime in which reduced solution-phase control leads to greater selectivity and higher sensitivity. Incorporation of dual-pH evaluation during peptide method development therefore provides a simple strategy for maximizing sensitivity and avoiding systematic bias in targeted LC\u2013MS\/MS assays.<\/jats:p>","DOI":"10.1021\/acs.analchem.6c02596","type":"journal-article","created":{"date-parts":[[2026,7,21]],"date-time":"2026-07-21T17:44:42Z","timestamp":1784655882000},"page":"22309-22317","source":"Crossref","is-referenced-by-count":0,"title":["Dual-pH LC\u2013ESI\u2013MS\/MS\nExpands Peptide\nDetectability via Wrong-Way-Round Ionization"],"prefix":"10.1021","volume":"98","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-1353-9633","authenticated-orcid":true,"given":"Ola","family":"Svahn","sequence":"first","affiliation":[{"name":"Kristianstad University , , ,","place":["Kristianstad, Sweden, SE-291 88"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jonatan","family":"Svahn","sequence":"additional","affiliation":[{"name":"Lund University , , ,","place":["Lund, Sweden, SE-221 00"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jan","family":"Astermark","sequence":"additional","affiliation":[{"name":"Sk\u00e5ne University Hospital , , ,","place":["Malmo\u0308, Sweden, SE-205 02"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2026,7,21]]},"reference":[{"issue":"1\u20132","key":"2026080408074001200_cit1","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1016\/j.jchromb.2008.07.021","article-title":"Quantitative bioanalysis of peptides by liquid chromatography\ncoupled to (tandem) mass spectrometry","volume":"872","author":"van den Broek","year":"2008","journal-title":"J. Chromatogr.\nB:Anal. Technol. Biomed. Life Sci."},{"issue":"6","key":"2026080408074001200_cit2","doi-asserted-by":"publisher","first-page":"362","DOI":"10.1002\/mas.10008","article-title":"Practical implications\nof some recent studies in electrospray\nionization fundamentals","volume":"20","author":"Cech","year":"2001","journal-title":"Mass Spectrom. Rev."},{"issue":"3","key":"2026080408074001200_cit3","doi-asserted-by":"publisher","first-page":"461","DOI":"10.1007\/s13361-016-1563-1","article-title":"pH Effects\non Electrospray Ionization\nEfficiency","volume":"28","author":"Liigand","year":"2017","journal-title":"J. Am. Soc. Mass Spectrom."},{"issue":"10","key":"2026080408074001200_cit4","doi-asserted-by":"publisher","first-page":"1143","DOI":"10.1002\/oms.1210271028","article-title":"Electrospray analysis\nof proteins: A comparison of positive-ion and negative-ion mass spectra\nat high and low pH","volume":"27","author":"Kelly","year":"1992","journal-title":"Org. Mass Spectrom."},{"issue":"8","key":"2026080408074001200_cit5","doi-asserted-by":"publisher","first-page":"419","DOI":"10.1002\/oms.1210290805","article-title":"Disparity\nbetween solution-phase equilibria and charge\nstate distributions in positive-ion electrospray mass spectrometry","volume":"29","author":"Wang","year":"1994","journal-title":"Org. Mass Spectrom."},{"issue":"9","key":"2026080408074001200_cit6","doi-asserted-by":"publisher","first-page":"847","DOI":"10.1093\/chromsci\/bmz061","article-title":"Effect of Mobile Phase pH on the\nElectrospray Ionization Efficiency and Qualitative Analysis of Pharmaceuticals\nin ESI + LC-MS\/MS","volume":"57","author":"Kafeenah","year":"2019","journal-title":"J. Chromatogr Sci."},{"key":"2026080408074001200_cit7","doi-asserted-by":"publisher","first-page":"128","DOI":"10.1016\/j.jchromb.2016.07.015","article-title":"Increased electrospray ionization intensities and expanded\nchromatographic possibilities for emerging contaminants using mobile\nphases of different pH","volume":"1033\u20131034","author":"Svahn","year":"2016","journal-title":"J. Chromatogr. B:Anal.\nTechnol. Biomed. Life Sci."},{"issue":"10","key":"2026080408074001200_cit8","doi-asserted-by":"publisher","first-page":"1120","DOI":"10.1002\/(sici)1097-0231(19970630)11:10&lt;1120:Aid-rcm976&gt;3.0.Co;2-q","article-title":"\u2019Wrong-way-round\u2019\nElectrospray\nIonization of Amino Acids","volume":"11","author":"Mansoori","year":"1997","journal-title":"Rapid Commun. Mass\nSpectrom."},{"issue":"22","key":"2026080408074001200_cit9","doi-asserted-by":"publisher","first-page":"972A","DOI":"10.1021\/ac00070a001","article-title":"From ions in solution\nto ions in the gas phase - the mechanism of\nelectrospray mass spectrometry","volume":"65","author":"Kebarle","year":"1993","journal-title":"Anal. Chem."},{"issue":"1","key":"2026080408074001200_cit10","doi-asserted-by":"publisher","first-page":"104","DOI":"10.1021\/jp209344z","article-title":"Modeling the Behavior of Coarse-Grained Polymer Chains\nin Charged Water Droplets: Implications for the Mechanism of Electrospray\nIonization","volume":"116","author":"Ahadi","year":"2012","journal-title":"J. Phys. Chem. B"},{"issue":"1","key":"2026080408074001200_cit11","doi-asserted-by":"publisher","first-page":"2","DOI":"10.1021\/ac302789c","article-title":"Unraveling the mechanism\nof electrospray ionization","volume":"85","author":"Konermann","year":"2013","journal-title":"Anal. Chem."},{"issue":"7","key":"2026080408074001200_cit12","doi-asserted-by":"publisher","first-page":"524","DOI":"10.1016\/1044-0305(93)85014-O","article-title":"Ion formation\nfrom charged droplets: roles of geometry, energy, and time","volume":"4","author":"Fenn","year":"1993","journal-title":"J. Am. Soc. Mass Spectrom."},{"issue":"4926","key":"2026080408074001200_cit13","doi-asserted-by":"publisher","first-page":"64","DOI":"10.1126\/science.2675315","article-title":"Electrospray\nIonization for Mass Spectrometry of Large Biomolecules","volume":"246","author":"Fenn","year":"1989","journal-title":"Science"},{"issue":"9824","key":"2026080408074001200_cit14","doi-asserted-by":"publisher","first-page":"1447","DOI":"10.1016\/S0140-6736(11)61139-2","article-title":"Modern haemophilia\ncare","volume":"379","author":"Berntorp","year":"2012","journal-title":"Lancet"},{"issue":"2","key":"2026080408074001200_cit15","doi-asserted-by":"publisher","first-page":"342","DOI":"10.1016\/j.jpba.2007.02.006","article-title":"Recent advances in high-throughput\nquantitative bioanalysis by LC-MS\/MS","volume":"44","author":"Xu","year":"2007","journal-title":"J. Pharm.\nBiomed. Anal."},{"issue":"2","key":"2026080408074001200_cit16","doi-asserted-by":"publisher","first-page":"137","DOI":"10.1586\/14789450.2014.877346","article-title":"MRM for\nthe verification\nof cancer biomarker proteins: recent applications to human plasma\nand serum","volume":"11","author":"Chambers","year":"2014","journal-title":"Expert Rev. Proteomics"},{"key":"2026080408074001200_cit17","doi-asserted-by":"publisher","DOI":"10.1016\/j.jpba.2019.112781","article-title":"Quantification\nof coagulation factor VIII in human plasma with liquid chromatography\ntandem mass spectrometry using a selective sample purification with\ncamelid nanobodies","volume":"175","author":"El Amrani","year":"2019","journal-title":"J. Pharm. Biomed. Anal"},{"issue":"19","key":"2026080408074001200_cit18","doi-asserted-by":"publisher","first-page":"1203","DOI":"10.4155\/bio-2023-0129","article-title":"Quantitative bioanalysis of proteins\nby digestion and LC-MS\/MS: the use of multiple signature peptides","volume":"15","author":"Sleumer","year":"2023","journal-title":"Bioanalysis"},{"issue":"7","key":"2026080408074001200_cit19","doi-asserted-by":"publisher","first-page":"966","DOI":"10.1093\/bioinformatics\/btq054","article-title":"Skyline: an open\nsource document editor for creating\nand analyzing targeted proteomics experiments","volume":"26","author":"MacLean","year":"2010","journal-title":"Bioinformatics"},{"key":"2026080408074001200_cit20","doi-asserted-by":"publisher","DOI":"10.1016\/j.jpba.2025.116879","article-title":"Alcohol-based solvents\nas mobile\nphases for LC-MS characterization of therapeutic proteins","volume":"262","author":"Zarei","year":"2025","journal-title":"J. Pharm. Biomed. Anal."},{"issue":"5","key":"2026080408074001200_cit21","first-page":"18","article-title":"Practical\nconsiderations in analysing biologically active peptides by Electrospray\nIonisation (ESI) Mass Spectrometry","volume":"18","author":"Nielsen","year":"2013","journal-title":"Eur. Biopharm.\nRev."},{"key":"2026080408074001200_cit22","doi-asserted-by":"publisher","first-page":"3715","DOI":"10.1016\/j.csbj.2023.07.027","article-title":"Insight on\nphysicochemical properties governing peptide MS1 response in HPLC-ESI-MS\/MS:\nA deep learning approach","volume":"21","author":"Abdul-Khalek","year":"2023","journal-title":"Comput. Struct. Biotechnol.\nJ."},{"issue":"23","key":"2026080408074001200_cit23","doi-asserted-by":"publisher","first-page":"4885","DOI":"10.1021\/ac970095w","article-title":"A Predictive\nModel for Matrix and Analyte Effects in Electrospray Ionization of\nSingly-Charged Ionic Analytes","volume":"69","author":"Enke","year":"1997","journal-title":"Anal. Chem."},{"issue":"4","key":"2026080408074001200_cit24","doi-asserted-by":"publisher","first-page":"1111","DOI":"10.1073\/pnas.0609969104","article-title":"Gas-phase ions of\nsolute species from charged droplets\nof solutions","volume":"104","author":"Nguyen","year":"2007","journal-title":"Proc. Natl. Acad. Sci. 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Mass\nSpectrom."},{"issue":"13","key":"2026080408074001200_cit27","doi-asserted-by":"publisher","first-page":"2045","DOI":"10.1182\/blood-2014-08-535328","article-title":"FVIII inhibitors:\npathogenesis and avoidance","volume":"125","author":"Astermark","year":"2015","journal-title":"Blood"}],"container-title":["Analytical Chemistry"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/pubs.acs.org\/doi\/pdf\/10.1021\/acs.analchem.6c02596","content-type":"application\/pdf","content-version":"vor","intended-application":"unspecified"},{"URL":"https:\/\/pubs.acs.org\/ancham\/article-pdf\/98\/30\/22309\/66090653\/acs.analchem.6c02596.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/pubs.acs.org\/ancham\/article-pdf\/98\/30\/22309\/66090653\/acs.analchem.6c02596.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,8,4]],"date-time":"2026-08-04T12:27:38Z","timestamp":1785846458000},"score":1,"resource":{"primary":{"URL":"https:\/\/pubs.acs.org\/ancham\/article\/98\/30\/22309\/5224856\/Dual-pH-LC-ESI-MS-MS-Expands-Peptide-Detectability"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,7,21]]},"references-count":27,"journal-issue":{"issue":"30","published-online":{"date-parts":[[2026,7,21]]},"published-print":{"date-parts":[[2026,8,4]]}},"URL":"https:\/\/doi.org\/10.1021\/acs.analchem.6c02596","relation":{},"ISSN":["0003-2700","1520-6882"],"issn-type":[{"value":"0003-2700","type":"print"},{"value":"1520-6882","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,7,21]]}}}