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They are cold-shock proteins CspC and CspE, DNA-binding proteins HU-\u03b1 and HU-\u03b2 and CsgA (the major subunit of curli). The fragmentation efficiency of these singly charged protein ions varied considerably. The pattern of fragment ions obtained from tandem mass spectrometry postsource decay of these protein ions was compared to in silico protein structures obtained using AlphaFold3 to better understand the factors that contribute to their gas phase dissociation as well as their likelihood of unfolding during MALDI sample preparation. Molecular dynamic simulations were performed to calculate the root-mean-square fluctuations (RMSF) of C\u03b1 atoms and root-mean-square deviation (RMSD) of the protein backbone to assess their propensity to unfold\/denature during MALDI sample preparation. A molecular dynamics simulation was performed on a curli multimer composed of one CsgB (the minor curli subunit) and five CsgA which showed rapid disintegration of this short curli complex in hexafluoro-isopropanol (HFIP) consistent with experimental observations. Strong hydrogen bonding of HFIP appears to disrupt the intermolecular hydrogen bonds of this amyloid protein complex but does not unfold CsgA monomer because of 138 intramolecular hydrogen bonds present in its solenoidal \u03b2-sheet structure. Finally, electrostatic analysis of the curli complex reveals partitioned regions of positive and negative charge along the fibril axis that may facilitate its assembly.<\/jats:p>","DOI":"10.1021\/jasms.6c00188","type":"journal-article","created":{"date-parts":[[2026,8,18]],"date-time":"2026-08-18T16:26:39Z","timestamp":1787070399000},"page":"2155-2167","source":"Crossref","is-referenced-by-count":0,"title":["Computational Simulations\nof Biofilm-Associated Proteins\nGenerated by Pathogenic\n                    <i>Escherichia coli<\/i>\n                    Identified by MALDI-TOF-TOF Mass Spectrometry and Top-Down Protein\nAnalysis"],"prefix":"10.1021","volume":"37","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-5787-1972","authenticated-orcid":true,"given":"Mahesh","family":"Koirala","sequence":"first","affiliation":[{"name":"Produce Safety & Microbiology, Western Regional Research Center, Agricultural Research Service , , , ,","place":["Albany, California, United States, 94710"]},{"name":"Research Participation Program Administered by the Oak Ridge Institute for Science and Education , , , ,","place":["Oak Ridge, Tennessee, United States, 37831-0117"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yanlin","family":"Shi","sequence":"additional","affiliation":[{"name":"Produce Safety & Microbiology, Western Regional Research Center, Agricultural Research Service , , , ,","place":["Albany, California, United States, 94710"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Michelle Q.","family":"Carter","sequence":"additional","affiliation":[{"name":"Produce Safety & Microbiology, Western Regional Research Center, Agricultural Research Service , , , ,","place":["Albany, California, United States, 94710"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2598-3686","authenticated-orcid":true,"given":"Clifton K.","family":"Fagerquist","sequence":"additional","affiliation":[{"name":"Produce Safety & Microbiology, Western Regional Research Center, Agricultural Research Service , , , ,","place":["Albany, California, United States, 94710"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2026,8,18]]},"reference":[{"issue":"6","key":"2026090311510934600_cit1","doi-asserted-by":"publisher","first-page":"598","DOI":"10.1016\/S1369-5274(99)00028-4","article-title":"Genetic analyses of\nbacterial biofilm formation","volume":"2","author":"Pratt","year":"1999","journal-title":"Curr. Opin. Microbiol."},{"key":"2026090311510934600_cit2","doi-asserted-by":"publisher","first-page":"131","DOI":"10.1146\/annurev.micro.60.080805.142106","article-title":"Curli biogenesis\nand function","volume":"60","author":"Barnhart","year":"2006","journal-title":"Annu. Rev. Microbiol."},{"issue":"1","key":"2026090311510934600_cit3","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-023-38204-2","article-title":"Structural analysis\nand architectural principles of the bacterial amyloid curli","volume":"14","author":"Sleutel","year":"2023","journal-title":"Nat. Commun."},{"issue":"10","key":"2026090311510934600_cit4","doi-asserted-by":"publisher","first-page":"954","DOI":"10.1016\/j.bbapap.2019.05.010","article-title":"Bacterial functional amyloids: Order\nfrom disorder","volume":"1867","author":"Jain","year":"2019","journal-title":"Biochim. Biophys. Acta Proteins\nProteomics"},{"issue":"8","key":"2026090311510934600_cit5","doi-asserted-by":"publisher","first-page":"1551","DOI":"10.1016\/j.bbamcr.2013.09.010","article-title":"Curli biogenesis: order out of disorder","volume":"1843","author":"Evans","year":"2014","journal-title":"Biochim. Biophys. Acta"},{"key":"2026090311510934600_cit6","doi-asserted-by":"publisher","first-page":"3685","DOI":"10.1128\/AEM.02315-10","article-title":"Distinct acid resistance\nand survival fitness displayed\nby curli variants of enterohemorrhagic Escherichia coli O157:H7","volume":"77","author":"Carter","year":"2011","journal-title":"Appl. Environ.\nMicrobiol."},{"issue":"21","key":"2026090311510934600_cit7","doi-asserted-by":"publisher","first-page":"7706","DOI":"10.1128\/AEM.02157-12","article-title":"RcsB contributes\nto the distinct stress fitness among Escherichia coli O157:H7 curli variants of the 1993\nhamburger-associated outbreak strains","volume":"78","author":"Carter","year":"2012","journal-title":"Appl.\nEnviron. Microbiol."},{"key":"2026090311510934600_cit8","doi-asserted-by":"publisher","DOI":"10.1016\/j.coelec.2026.101832","article-title":"Probing the\nInner World of Microbial Cities: Electrochemical Sensors for Characterizing\nBiofilm Chemical Microenvironments","volume":"57","author":"Koley","year":"2026","journal-title":"Curr. Opin.\nElectrochem."},{"issue":"2","key":"2026090311510934600_cit9","doi-asserted-by":"publisher","first-page":"183","DOI":"10.1002\/jemt.70064","article-title":"Biofilm Analysis\nby Confocal Microscopy-Basics and\nPractical Aspects","volume":"89","author":"Neu","year":"2026","journal-title":"Microsc. Res. 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Biotechnol."},{"issue":"4","key":"2026090311510934600_cit13","doi-asserted-by":"publisher","DOI":"10.3390\/v12040407","article-title":"The application of impedance\nspectroscopy for Pseudomonas biofilm monitoring during\nphage infection","volume":"12","author":"Gu\u0142a","year":"2020","journal-title":"Viruses"},{"key":"2026090311510934600_cit14","doi-asserted-by":"publisher","first-page":"99","DOI":"10.1016\/S0003-2670(00)82721-X","article-title":"The oscillation frequency of a quartz\nresonator in contact with liquid","volume":"175","author":"Keiji Kanazawa","year":"1985","journal-title":"Anal. Chim.\nActa"},{"key":"2026090311510934600_cit15","doi-asserted-by":"publisher","first-page":"313","DOI":"10.1016\/j.bios.2008.04.011","article-title":"An interleukin-6\nZnO\/SiO2\/Si surface acoustic wave biosensor","volume":"24","author":"Krishnamoorthy","year":"2008","journal-title":"Biosens. Bioelectron."},{"key":"2026090311510934600_cit16","doi-asserted-by":"publisher","first-page":"136","DOI":"10.1016\/j.snb.2012.01.021","article-title":"An\nALD aluminum oxide\npassivated surface acoustic wave sensor for early biofilm detection","volume":"163","author":"Kim","year":"2012","journal-title":"Sens. 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Dairy Sci."},{"issue":"5","key":"2026090311510934600_cit19","doi-asserted-by":"publisher","first-page":"2148","DOI":"10.1111\/jam.15049","article-title":"Diagnosis\nof biofilm infections:\ncurrent methods used, challenges and perspectives for the future","volume":"131","author":"Silva","year":"2021","journal-title":"J. Appl. Microbiol."},{"key":"2026090311510934600_cit20","unstructured":"Fagerquist, C. K.; Shi, Y. In  Biofilm\nof Pathogenic Bacteria Analyzed by MALDI-TOF Imaging Mass Spectrometry\n(IMS) and Top-Down Proteomic Identification. Proceedings\nof the 71st ASMS Conference on Mass Spectrometry and Allied Topics.\n(TP 330)."},{"issue":"10","key":"2026090311510934600_cit21","doi-asserted-by":"publisher","first-page":"1241","DOI":"10.1002\/rcm.6220","article-title":"A new calibrant for\nmatrix-assisted laser desorption\/ionization\ntime-of-flight-time-of-flight post-source decay tandem mass spectrometry\nof non-digested proteins for top-down proteomic analysis","volume":"26","author":"Fagerquist","year":"2012","journal-title":"Rapid Commun. 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Protein Crystallogr."},{"key":"2026090311510934600_cit28","doi-asserted-by":"publisher","first-page":"19","DOI":"10.1016\/j.softx.2015.06.001","article-title":"Gromacs: High performance\nmolecular simulations through multi-level parallelism from laptops\nto supercomputers","volume":"1\u20132","author":"Abraham","year":"2015","journal-title":"SoftwareX"},{"issue":"suppl_2","key":"2026090311510934600_cit29","doi-asserted-by":"publisher","first-page":"W368","DOI":"10.1093\/nar\/gki464","article-title":"H++: a server for estimating pKas and adding\nmissing hydrogens to macromolecules","volume":"33","author":"Gordon","year":"2005","journal-title":"Nucleic\nAcids Res."},{"issue":"25","key":"2026090311510934600_cit30","doi-asserted-by":"publisher","first-page":"2135","DOI":"10.1002\/jcc.23354","article-title":"CHARMM36 all-atom additive protein\nforce field: Validation based on comparison to NMR data","volume":"34","author":"Huang","year":"2013","journal-title":"J. Comput. Chem."},{"issue":"11","key":"2026090311510934600_cit31","doi-asserted-by":"publisher","first-page":"5834","DOI":"10.1021\/acs.jctc.8b00768","article-title":"Automated topology\nbuilder version 3.0: Prediction\nof solvation free enthalpies in water and hexane","volume":"14","author":"Stroet","year":"2018","journal-title":"J. Chem. Theory Comput."},{"issue":"1","key":"2026090311510934600_cit32","doi-asserted-by":"publisher","DOI":"10.1063\/1.2408420","article-title":"Canonical\nsampling through velocity\nrescaling","volume":"126","author":"Bussi","year":"2007","journal-title":"J. Chem. Phys."},{"issue":"12","key":"2026090311510934600_cit33","doi-asserted-by":"publisher","first-page":"7182","DOI":"10.1063\/1.328693","article-title":"Polymorphic transitions in single crystals: A new molecular\ndynamics method","volume":"52","author":"Parrinello","year":"1981","journal-title":"J. Appl. Phys."},{"issue":"6","key":"2026090311510934600_cit34","doi-asserted-by":"publisher","DOI":"10.3390\/molecules30061277","article-title":"Binding\nfree energy analysis of colicin D, E3 and E8\nto their respective cognate immunity proteins using computational\nsimulations","volume":"30","author":"Koirala","year":"2025","journal-title":"Molecules"},{"issue":"1","key":"2026090311510934600_cit35","doi-asserted-by":"publisher","DOI":"10.1038\/s41598-017-09466-w","article-title":"Dynamics of Hydration\nWater Plays a Key Role in Determining the Binding\nThermodynamics of Protein Complexes","volume":"7","author":"Chong","year":"2017","journal-title":"Sci. Rep."},{"issue":"35","key":"2026090311510934600_cit36","doi-asserted-by":"publisher","first-page":"11920","DOI":"10.1021\/acs.analchem.0c05480","article-title":"A Caffeic\nAcid Matrix Improves In Situ Detection and Imaging of Proteins with\nHigh Molecular Weight Close to 200,000 Da in Tissues by Matrix-Assisted\nLaser Desorption\/Ionization Mass Spectrometry Imaging","volume":"93","author":"Liu","year":"2021","journal-title":"Anal. Chem."},{"issue":"8","key":"2026090311510934600_cit37","doi-asserted-by":"publisher","first-page":"1739","DOI":"10.1039\/c0an00909a","article-title":"Induction and identification\nof disulfide-intact and\ndisulfide-reduced \u03b2-subunit of Shiga toxin 2 from Escherichia coli O157:H7 using MALDI-TOF-TOF-MS\/MS\nand top-down proteomics","volume":"136","author":"Fagerquist","year":"2011","journal-title":"Analyst"},{"issue":"8","key":"2026090311510934600_cit38","doi-asserted-by":"publisher","DOI":"10.1002\/rcm.70031","article-title":"Colicin-Bearing Plasmids Carried\nby Shiga Toxin-Producing E. coli (STEC)\nAnalyzed by Targeted Top-Down MS Analysis","volume":"40","author":"Fagerquist","year":"2026","journal-title":"Rapid\nCommun. Mass Spectrom."},{"issue":"5","key":"2026090311510934600_cit39","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0299287","article-title":"Exploring\nthe fragmentation efficiency of proteins\nanalyzed by MALDI-TOF-TOF tandem mass spectrometry using computational\nand statistical analyses","volume":"19","author":"Park","year":"2024","journal-title":"PLoS One"},{"key":"2026090311510934600_cit40","doi-asserted-by":"publisher","DOI":"10.1016\/j.ijms.2021.116736","article-title":"Sequestration of\nthe ionizing proton in singly charged\nmetastable protein ions generated by MALDI","volume":"471","author":"Fagerquist","year":"2022","journal-title":"Int. J. Mass Spectrom."},{"key":"2026090311510934600_cit41","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1016\/j.ijms.2018.12.006","article-title":"Software for\ntop-down proteomic identification of a plasmid-borne factor (and other\nproteins) from genomically sequenced pathogenic bacteria using MALDI-TOF-TOF-MS\/MS\nand post-source decay","volume":"438","author":"Fagerquist","year":"2019","journal-title":"Int. J. Mass Spectrom."},{"issue":"11","key":"2026090311510934600_cit42","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0260650","article-title":"Top-down proteomic\nidentification of plasmid and host\nproteins produced by pathogenic Escherichia coli using MALDI-TOF-TOF tandem mass spectrometry","volume":"16","author":"Fagerquist","year":"2021","journal-title":"PLoS One"},{"key":"2026090311510934600_cit43","doi-asserted-by":"publisher","first-page":"815","DOI":"10.1016\/j.mex.2019.04.011","article-title":"Top-down and middle-down proteomic\nanalysis of Shiga\ntoxin using MALDI-TOF-TOF mass spectrometry","volume":"6","author":"Fagerquist","year":"2019","journal-title":"MethodsX"},{"issue":"4","key":"2026090311510934600_cit44","doi-asserted-by":"publisher","DOI":"10.1128\/msystems.00086-22","article-title":"Cellular RNA Targets of Cold Shock\nProteins CspC and CspE and Their Importance for Serum Resistance in\nSepticemic Escherichia coli","volume":"7","author":"Yair","year":"2022","journal-title":"mSystems"},{"key":"2026090311510934600_cit45","doi-asserted-by":"publisher","DOI":"10.1038\/ncomms5945","article-title":"Programmable biofilm-based\nmaterials from engineered curli nanofibres","volume":"5","author":"Nguyen","year":"2014","journal-title":"Nat. Commun."},{"key":"2026090311510934600_cit46","doi-asserted-by":"publisher","first-page":"53","DOI":"10.1007\/978-1-62703-245-2_4","article-title":"Experimental manipulation\nof the microbial functional amyloid called curli","volume":"966","author":"Zhou","year":"2013","journal-title":"Methods Mol. Biol."}],"container-title":["Journal of the American\nSociety for Mass Spectrometry"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/pubs.acs.org\/doi\/pdf\/10.1021\/jasms.6c00188","content-type":"application\/pdf","content-version":"vor","intended-application":"unspecified"},{"URL":"https:\/\/pubs.acs.org\/jamsef\/article-pdf\/37\/9\/2155\/66834093\/jasms.6c00188.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/pubs.acs.org\/jamsef\/article-pdf\/37\/9\/2155\/66834093\/jasms.6c00188.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,9,3]],"date-time":"2026-09-03T16:23:22Z","timestamp":1788452602000},"score":1,"resource":{"primary":{"URL":"https:\/\/pubs.acs.org\/jamsef\/article\/37\/9\/2155\/5274504\/Computational-Simulations-of-Biofilm-Associated"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,8,18]]},"references-count":46,"journal-issue":{"issue":"9","published-online":{"date-parts":[[2026,8,18]]},"published-print":{"date-parts":[[2026,9,2]]}},"URL":"https:\/\/doi.org\/10.1021\/jasms.6c00188","relation":{},"ISSN":["1044-0305","1879-1123"],"issn-type":[{"value":"1044-0305","type":"print"},{"value":"1879-1123","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,8,18]]}}}