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Previous studies identify some sites of isomerization and racemization of deamidated asparaginyl and aspartyl residues in \u0263S but have been limited due to the complexity of isoforms and difficulty in characterizing deamidation posttranslational modifications. A total of 32 stable isotope\u2010labeled peptides are created for \u0263S residues 7\u201318, 72\u201378, and 131\u2013145, containing L\u2010Asp, D\u2010Asp, L\u2010isoAsp, and D\u2010isoAsp at D12, N14, N76, D77, and N143 to act as internal chromatography standards spiked into tryptic digests of nuclear insoluble protein of a cataractous human lens. High\u2010resolution mass spectrometry is used to accurately assign deamidation status using the 19\u2009mDa mass defect between isotopic peaks of deamidated and nondeamidated peptides. While peptides containing D\u2010forms of Asp and isoAsp were assigned, the predominant isoforms contained L\u2010isoAsp. High\u2010resolution mass spectrometry using wide single ion monitoring data\u2010independent acquisition also greatly improved the reliable identification of peptide deamidation states. These results will aid creation of \u0263S using native chemical ligation to examine the role of isoAsp in crystallin aggregation and cataract.<\/jats:p>","DOI":"10.1002\/cbic.202500352","type":"journal-article","created":{"date-parts":[[2025,7,14]],"date-time":"2025-07-14T08:54:16Z","timestamp":1752483256000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Isomerized and Racemized Aspartyl and Deamidated Asparagine Residues Identified in \u0263S\u2010Crystallin"],"prefix":"10.1002","volume":"26","author":[{"given":"Victoria S.","family":"Halls","sequence":"first","affiliation":[{"name":"Medicinal Chemistry Shared Resource University Shared Resources Oregon Health &amp; Science University  3181 SW Sam Jackson Park Rd Portland OR 97239 USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0005-4838-284X","authenticated-orcid":false,"given":"Larry L.","family":"David","sequence":"additional","affiliation":[{"name":"Department of Chemical Physiology and Biochemistry School of Medicine Oregon Health &amp; Science University  3181 SW Sam Jackson Park Rd Portland OR 97239 USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Keith D.","family":"Zientek","sequence":"additional","affiliation":[{"name":"Proteomics Shared Resource University Shared Resources Oregon Health &amp; Science University  3181 SW Sam Jackson Park Rd Portland OR 97239 USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7906-6699","authenticated-orcid":false,"given":"Kirsten J.","family":"Lampi","sequence":"additional","affiliation":[{"name":"Department of Biomaterials and Biomedical Sciences School of Dentistry Oregon Health &amp; Science University  Robertson Collaborative Life Sciences Building &amp; Skourtes Tower, 2730 S Moody Ave Portland OR 97239 USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2025,7,14]]},"reference":[{"key":"e_1_2_9_2_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.tibs.2016.06.004"},{"key":"e_1_2_9_3_1","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0306856"},{"key":"e_1_2_9_4_1","doi-asserted-by":"publisher","DOI":"10.1021\/acs.analchem.9b03127"},{"key":"e_1_2_9_5_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.bbagen.2015.08.001"},{"key":"e_1_2_9_6_1","doi-asserted-by":"publisher","DOI":"10.1167\/iovs.11-9085"},{"key":"e_1_2_9_7_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.exer.2018.03.018"},{"key":"e_1_2_9_8_1","doi-asserted-by":"publisher","DOI":"10.1093\/braincomms\/fcab028"},{"key":"e_1_2_9_9_1","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.RA119.009052"},{"key":"e_1_2_9_10_1","doi-asserted-by":"publisher","DOI":"10.1076\/ceyr.22.2.148.5524"},{"key":"e_1_2_9_11_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.chroma.2016.07.021"},{"key":"e_1_2_9_12_1","doi-asserted-by":"publisher","DOI":"10.1021\/acs.analchem.8b04696"},{"key":"e_1_2_9_13_1","doi-asserted-by":"publisher","DOI":"10.1021\/ac504413e"},{"key":"e_1_2_9_14_1","doi-asserted-by":"publisher","DOI":"10.1016\/bs.mie.2019.06.020"},{"key":"e_1_2_9_15_1","doi-asserted-by":"publisher","DOI":"10.1021\/ac101806e"},{"key":"e_1_2_9_16_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.jasms.2005.08.019"},{"key":"e_1_2_9_17_1","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M112.399972"},{"key":"e_1_2_9_18_1","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.RA118.007052"},{"key":"e_1_2_9_19_1","doi-asserted-by":"publisher","DOI":"10.1186\/s40478-016-0348-x"},{"key":"e_1_2_9_20_1","first-page":"1211","volume":"11","author":"Robinson N. 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