{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,31]],"date-time":"2026-08-31T23:16:15Z","timestamp":1788218175874,"version":"build-2803163510"},"reference-count":29,"publisher":"American Chemical Society (ACS)","issue":"33","license":[{"start":{"date-parts":[[2026,8,13]],"date-time":"2026-08-13T00:00:00Z","timestamp":1786579200000},"content-version":"stm-asf","delay-in-days":0,"URL":"https:\/\/doi.org\/10.15223\/policy-029"},{"start":{"date-parts":[[2026,8,13]],"date-time":"2026-08-13T00:00:00Z","timestamp":1786579200000},"content-version":"stm-asf","delay-in-days":0,"URL":"https:\/\/doi.org\/10.15223\/policy-037"},{"start":{"date-parts":[[2026,8,13]],"date-time":"2026-08-13T00:00:00Z","timestamp":1786579200000},"content-version":"stm-asf","delay-in-days":0,"URL":"https:\/\/doi.org\/10.15223\/policy-045"}],"funder":[{"DOI":"10.13039\/100000001","name":"National Science Foundation","doi-asserted-by":"publisher","award":["32141003, 32371503"],"award-info":[{"award-number":["32141003, 32371503"]}],"id":[{"id":"10.13039\/100000001","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/na","name":"CAMS Innovation Fund for Medical Sciences","doi-asserted-by":"publisher","award":["2019-I2M5-017, 2022-I2M-C&T-B-082"],"award-info":[{"award-number":["2019-I2M5-017, 2022-I2M-C&T-B-082"]}],"id":[{"id":"10.13039\/na","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/na","name":"National Basic Research Programs","doi-asserted-by":"publisher","award":["2022YFA1304600, 2024YFC3405400"],"award-info":[{"award-number":["2022YFA1304600, 2024YFC3405400"]}],"id":[{"id":"10.13039\/na","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/na","name":"Beijing Life Science Academy","doi-asserted-by":"publisher","award":["2024300CB0100"],"award-info":[{"award-number":["2024300CB0100"]}],"id":[{"id":"10.13039\/na","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/na","name":"Beijing Natural Science Foundation","doi-asserted-by":"publisher","award":["L246037"],"award-info":[{"award-number":["L246037"]}],"id":[{"id":"10.13039\/na","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/na","name":"Foundation of State Key Lab of Proteomics","doi-asserted-by":"publisher","award":["SKLP-O202201, SKLP-K202201"],"award-info":[{"award-number":["SKLP-O202201, SKLP-K202201"]}],"id":[{"id":"10.13039\/na","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2026,8,25]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>LysN is a valuable protease in proteomics because it cleaves peptide bonds N-terminal to lysine, generating peptides with physicochemical properties complementary to those produced by LysC and trypsin. However, the broader adoption of LysN in proteomic workflows has been limited by the lack of commercially available enzymes that combine high activity, low missed-cleavage rates, and sufficient stability under practical sample-processing conditions. Here, we report the recombinant production and proteomic characterization of a self-digestion-resistant and highly active LysN from Shewanella loihica (SL-LysN). Using terminomics, we mapped the mature N- and C-termini of the enzyme and established the primary structure of the active protease. We further developed a high-density fermentation, refolding, and purification workflow to obtain highly purified recombinant SL-LysN. Biochemical and proteomic benchmarking showed that SL-LysN displayed 3.3-fold higher specific activity than commercial LysN and reduced missed cleavages by approximately 80%. Notably, SL-LysN retained high activity in the presence of 8 M urea or 1% SDS and showed strong resistance to autolysis, indicating exceptional robustness for proteomic sample preparation. In complex mammalian proteome digests, SL-LysN achieved &amp;gt;95% cleavage specificity and a missed-cleavage rate of only 5.9%. These features address a long-standing bottleneck in N-terminal proteolysis and establish SL-LysN as a high-performance enzymatic tool for advanced proteomic workflows, including deep protein sequencing, quantitative proteomics, terminomics, de novo sequencing and analyses requiring efficient digestion under denaturing conditions.<\/jats:p>","DOI":"10.1021\/acs.analchem.6c02641","type":"journal-article","created":{"date-parts":[[2026,8,13]],"date-time":"2026-08-13T21:02:07Z","timestamp":1786654927000},"page":"24278-24289","source":"Crossref","is-referenced-by-count":0,"title":["A Robust, Self-Digestion-Resistant\nLysN with Superior\nActivity and Cleavage Fidelity for Advanced Proteomic Workflows"],"prefix":"10.1021","volume":"98","author":[{"given":"Jiaxing","family":"Dai","sequence":"first","affiliation":[{"name":"Beijing Life Science Academy , ,","place":["Beijing, China, 102209"]},{"name":"Beijing Proteome Research Center , ,","place":["Beijing, China, 102206"]},{"name":"Hebei University , , ,","place":["Baoding, China, 071002"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jiahui","family":"Shi","sequence":"additional","affiliation":[{"name":"Academy of Military Medical Sciences , , ,","place":["Beijing, China, 102206"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tao","family":"Zhang","sequence":"additional","affiliation":[{"name":"Academy of Military Medical Sciences , , ,","place":["Beijing, China, 102206"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lei","family":"Chang","sequence":"additional","affiliation":[{"name":"Beijing Life Science Academy , ,","place":["Beijing, China, 102209"]},{"name":"Academy of Military Medical Sciences , , ,","place":["Beijing, China, 102206"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5834-2829","authenticated-orcid":true,"given":"Yao","family":"Zhang","sequence":"additional","affiliation":[{"name":"Beijing Life Science Academy , ,","place":["Beijing, China, 102209"]},{"name":"Academy of Military Medical Sciences , , ,","place":["Beijing, China, 102206"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yuping","family":"Xie","sequence":"additional","affiliation":[{"name":"Academy of Military Medical Sciences , , ,","place":["Beijing, China, 102206"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Bin","family":"Fu","sequence":"additional","affiliation":[{"name":"Academy of Military Medical Sciences , , ,","place":["Beijing, China, 102206"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhitang","family":"Lyu","sequence":"additional","affiliation":[{"name":"Hebei University , , ,","place":["Baoding, China, 071002"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0628-6981","authenticated-orcid":true,"given":"Ping","family":"Xu","sequence":"additional","affiliation":[{"name":"Beijing Life Science Academy , ,","place":["Beijing, China, 102209"]},{"name":"Academy of Military Medical Sciences , , ,","place":["Beijing, China, 102206"]},{"name":"Hebei University , , ,","place":["Baoding, China, 071002"]},{"name":"China Medical University , , ,","place":["Shenyang, China, 110122"]},{"name":"Beijing Normal University , , ,","place":["Beijing, China, 100875"]},{"name":"Hunan University , , ,","place":["Changsha, China, 410082"]},{"name":"University of South China , , ,","place":["Hengyang, China, 421001"]},{"name":"Anhui Medical University , , ,","place":["Hefei, China, 230032"]},{"name":"Guizhou University Medical College , ,","place":["Guiyang, China, 550025"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2026,8,13]]},"reference":[{"issue":"6","key":"2026083118455129400_cit1","doi-asserted-by":"publisher","first-page":"608","DOI":"10.1074\/mcp.T400003-MCP200","article-title":"Trypsin Cleaves Exclusively C-Terminal\nto Arginine and Lysine Residues","volume":"3","author":"Olsen","year":"2004","journal-title":"Mol. Cell.\nProteomics"},{"issue":"14","key":"2026083118455129400_cit2","doi-asserted-by":"publisher","first-page":"2612","DOI":"10.1111\/febs.13287","article-title":"Proteomics beyond\nTrypsin","volume":"282","author":"Tsiatsiani","year":"2015","journal-title":"FEBS\nJ."},{"issue":"3","key":"2026083118455129400_cit3","doi-asserted-by":"publisher","first-page":"1323","DOI":"10.1021\/pr900863u","article-title":"Value of\nUsing Multiple Proteases\nfor Large-Scale Mass Spectrometry-Based Proteomics","volume":"9","author":"Swaney","year":"2010","journal-title":"J. Proteome Res."},{"issue":"12","key":"2026083118455129400_cit4","doi-asserted-by":"publisher","first-page":"4352","DOI":"10.1021\/acs.jproteome.7b00340","article-title":"Multi-Protease\nStrategy Identifies Three PE2Missing Proteins in Human Testis Tissue","volume":"16","author":"Wang","year":"2017","journal-title":"J. Proteome Res."},{"issue":"3","key":"2026083118455129400_cit5","doi-asserted-by":"publisher","first-page":"485","DOI":"10.1002\/pmic.201000537","article-title":"Accurate Mass Comparison\nCoupled with Two Endopeptidases Enables Identification of Protein\nTermini","volume":"11","author":"Kishimoto","year":"2011","journal-title":"Proteomics"},{"issue":"4","key":"2026083118455129400_cit6","doi-asserted-by":"publisher","first-page":"650","DOI":"10.1074\/mcp.m800249-mcp200","article-title":"Straightforward\nde\nNovo Peptide Sequencing by MALDI-MS\/MS Using a Lys-N Metalloendopeptidase","volume":"8","author":"Boersema","year":"2009","journal-title":"Mol. Cell. Proteomics"},{"issue":"5","key":"2026083118455129400_cit7","doi-asserted-by":"publisher","first-page":"405","DOI":"10.1038\/nmeth.1204","article-title":"Straightforward\nLadder\nSequencing of Peptides Using a Lys-N Metalloendopeptidase","volume":"5","author":"Taouatas","year":"2008","journal-title":"Nat. Methods"},{"issue":"1","key":"2026083118455129400_cit8","doi-asserted-by":"publisher","first-page":"55","DOI":"10.1038\/nmeth.3177","article-title":"LysargiNase Mirrors Trypsin for Protein C-Terminal\nand Methylation-Site Identification","volume":"12","author":"Huesgen","year":"2015","journal-title":"Nat. Methods"},{"issue":"24","key":"2026083118455129400_cit9","doi-asserted-by":"publisher","first-page":"15890","DOI":"10.1021\/acs.analchem.9b04340","article-title":"Ac-LysargiNase\nComplements Trypsin for the Identification of Ubiquitinated Sites","volume":"91","author":"Xiao","year":"2019","journal-title":"Anal. Chem."},{"issue":"17","key":"2026083118455129400_cit10","doi-asserted-by":"publisher","first-page":"1381","DOI":"10.1002\/rcm.8479","article-title":"LysargiNase Enhances Protein Identification on the\nBasis of Trypsin\non Formalin-Fixed Paraffin-Embedded Samples","volume":"33","author":"Liu","year":"2019","journal-title":"Rapid Commun. Mass Spectrom."},{"issue":"3","key":"2026083118455129400_cit11","doi-asserted-by":"publisher","first-page":"361","DOI":"10.1107\/S0907444900019740","article-title":"Structure of a New\nAspzincin Metalloendopeptidase from Grifola frondosa: Implications\nfor the Catalytic Mechanism and Substrate Specificity Based on Several\nDifferent Crystal Forms","volume":"57","author":"Hori","year":"2001","journal-title":"Acta Crystallogr. D\nBiol. Crystallogr."},{"issue":"4","key":"2026083118455129400_cit12","doi-asserted-by":"publisher","first-page":"461","DOI":"10.1093\/jb\/mvv115","article-title":"Heterologous Expression of Peptidyl-Lys\nMetallopeptidase of Armillaria mellea and Mutagenic Analysis of the\nRecombinant Peptidase","volume":"159","author":"\u00d8dum","year":"2016","journal-title":"J. Biochem."},{"issue":"2","key":"2026083118455129400_cit13","doi-asserted-by":"publisher","DOI":"10.1002\/rcm.8573","article-title":"Peptidyl-Lys Metalloendopeptidase\n(Lys-N) Purified from Dry Fruit of Grifola frondosa Demonstrates Mirror\nDigestion Property with Lysyl Endopeptidase (Lys-C)","volume":"34","author":"Zhao","year":"2020","journal-title":"Rapid Commun. Mass Spectrom."},{"issue":"46","key":"2026083118455129400_cit14","doi-asserted-by":"publisher","first-page":"8827","DOI":"10.1039\/c0cc02523b","article-title":"Cleavage Specificities\nof the Brother and Sister Proteases Lys-C and Lys-N","volume":"46","author":"Raijmakers","year":"2009","journal-title":"Chem. Commun."},{"issue":"D1","key":"2026083118455129400_cit15","doi-asserted-by":"publisher","first-page":"D624","DOI":"10.1093\/nar\/gkx1134","article-title":"The MEROPS Database of Proteolytic Enzymes, Their Substrates\nand Inhibitors in 2017 and a Comparison with Peptidases in the PANTHER\nDatabase","volume":"46","author":"Rawlings","year":"2018","journal-title":"Nucleic Acids Res."},{"issue":"4","key":"2026083118455129400_cit16","doi-asserted-by":"publisher","first-page":"1459","DOI":"10.1021\/acs.jproteome.9b00713","article-title":"Tryp-N:\nA Thermostable Protease for the Production of N-Terminal Argininyl\nand Lysinyl Peptides","volume":"19","author":"Wilson","year":"2020","journal-title":"J. Proteome Res."},{"key":"2026083118455129400_cit17","doi-asserted-by":"publisher","first-page":"120","DOI":"10.1016\/j.pep.2015.08.025","article-title":"Development\nof a Rapid High-Efficiency Scalable Process\nfor Acetylated Sus Scrofa Cationic Trypsin Production from Escherichia\nColi Inclusion Bodies","volume":"116","author":"Zhao","year":"2015","journal-title":"Protein Expr. Purif."},{"key":"2026083118455129400_cit18","doi-asserted-by":"publisher","first-page":"1015140","DOI":"10.3389\/fmicb.2022.1015140","article-title":"Mirror Proteases of Ac-Trypsin and Ac-LysargiNase Precisely\nImprove Novel Event Identifications in Mycolicibacterium Smegmatis\nMC2 155 by Proteogenomic Analysis","volume":"13","author":"Jiang","year":"2022","journal-title":"Front. Microbiol."},{"issue":"5","key":"2026083118455129400_cit19","doi-asserted-by":"publisher","first-page":"359","DOI":"10.1038\/nmeth.1322","article-title":"Universal\nSample Preparation\nMethod for Proteome Analysis","volume":"6","author":"Wis\u0301niewski","year":"2009","journal-title":"Nat. Methods"},{"issue":"15","key":"2026083118455129400_cit20","doi-asserted-by":"publisher","first-page":"2229","DOI":"10.1002\/pmic.201200591","article-title":"Systematic Research\non the Pretreatment of Peptides for Quantitative\nProteomics Using a C18 Microcolumn","volume":"13","author":"Zhai","year":"2013","journal-title":"Proteomics"},{"issue":"12","key":"2026083118455129400_cit21","doi-asserted-by":"publisher","first-page":"1067","DOI":"10.1002\/rcm.8440","article-title":"Recombinant Expression, Purification and Characterization of Acetylated\nLysargiNase from Escherichia coli with High Activity and Stability","volume":"33","author":"Zhang","year":"2019","journal-title":"Rapid Commun. Mass Spectrom."},{"issue":"3","key":"2026083118455129400_cit22","doi-asserted-by":"publisher","first-page":"746","DOI":"10.1053\/j.gastro.2023.05.039","article-title":"Macrophage-Derived\nCathepsin S Remodels the Extracellular Matrix to Promote Liver Fibrogenesis","volume":"165","author":"Zuo","year":"2023","journal-title":"Gastroenterology"},{"issue":"2","key":"2026083118455129400_cit23","doi-asserted-by":"publisher","first-page":"630","DOI":"10.1128\/iai.19.2.630-648.1978","article-title":"Purification of Pseudomonas Aeruginosa\nProteases and\nMicroscopic Characterization of Pseudomonal Protease-Induced Rabbit\nCorneal Damage","volume":"19","author":"Kreger","year":"1978","journal-title":"Infect. Immun."},{"issue":"11","key":"2026083118455129400_cit24","doi-asserted-by":"publisher","first-page":"1059","DOI":"10.1038\/nbt.4236","article-title":"Comprehensive\nIdentification of Peptides in Tandem Mass Spectra Using\nan Efficient Open Search Engine","volume":"36","author":"Chi","year":"2018","journal-title":"Nat. Biotechnol."},{"issue":"4","key":"2026083118455129400_cit25","doi-asserted-by":"publisher","first-page":"1454","DOI":"10.1016\/j.jprot.2011.11.016","article-title":"Systematic\nand Quantitative Comparison of Digest Efficiency and Specificity Reveals\nthe Impact of Trypsin Quality on MS-Based Proteomics","volume":"75","author":"Burkhart","year":"2012","journal-title":"J. Proteomics"},{"key":"2026083118455129400_cit26","doi-asserted-by":"publisher","first-page":"16","DOI":"10.1016\/j.jprot.2014.06.017","article-title":"Decreasing the Amount\nof Trypsin\nin In-Gel Digestion Leads to Diminished Chemical Noise and Improved\nProtein Identifications","volume":"109","author":"Hu","year":"2014","journal-title":"J. Proteomics"},{"key":"2026083118455129400_cit27","doi-asserted-by":"publisher","first-page":"127","DOI":"10.1007\/978-1-60327-310-7_9","article-title":"Protocol to Enrich and Analyze Plasma Membrane Proteins","volume-title":"Methods Mol. Biol.","author":"Wis\u0301niewski","year":"2009"},{"issue":"10","key":"2026083118455129400_cit28","doi-asserted-by":"publisher","first-page":"5422","DOI":"10.1021\/pr100656u","article-title":"A Quantitative Study\nof the Effects of Chaotropic Agents, Surfactants, and Solvents on\nthe Digestion Efficiency of Human Plasma Proteins by Trypsin","volume":"9","author":"Proc","year":"2010","journal-title":"J. Proteome Res."},{"issue":"D1","key":"2026083118455129400_cit29","doi-asserted-by":"publisher","first-page":"D1211","DOI":"10.1093\/nar\/gky869","article-title":"iProX: An Integrated Proteome Resource","volume":"47","author":"Ma","year":"2019","journal-title":"Nucleic Acids Res."}],"container-title":["Analytical Chemistry"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/pubs.acs.org\/doi\/pdf\/10.1021\/acs.analchem.6c02641","content-type":"application\/pdf","content-version":"vor","intended-application":"unspecified"},{"URL":"https:\/\/pubs.acs.org\/ancham\/article-pdf\/98\/33\/24278\/66537749\/acs.analchem.6c02641.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/pubs.acs.org\/ancham\/article-pdf\/98\/33\/24278\/66537749\/acs.analchem.6c02641.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,8,31]],"date-time":"2026-08-31T22:45:59Z","timestamp":1788216359000},"score":1,"resource":{"primary":{"URL":"https:\/\/pubs.acs.org\/ancham\/article\/98\/33\/24278\/5254947\/A-Robust-Self-Digestion-Resistant-LysN-with"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,8,13]]},"references-count":29,"journal-issue":{"issue":"33","published-online":{"date-parts":[[2026,8,13]]},"published-print":{"date-parts":[[2026,8,25]]}},"URL":"https:\/\/doi.org\/10.1021\/acs.analchem.6c02641","relation":{},"ISSN":["0003-2700","1520-6882"],"issn-type":[{"value":"0003-2700","type":"print"},{"value":"1520-6882","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,8,13]]}}}