{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,4,22]],"date-time":"2026-04-22T16:43:53Z","timestamp":1776876233596,"version":"3.51.2"},"reference-count":35,"publisher":"Wiley","issue":"8","license":[{"start":{"date-parts":[[2015,5,29]],"date-time":"2015-05-29T00:00:00Z","timestamp":1432857600000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/onlinelibrary.wiley.com\/termsAndConditions#vor"}],"funder":[{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"crossref","award":["GM69429"],"award-info":[{"award-number":["GM69429"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"crossref"}]},{"name":"National Sciences and Engineering Council of Canada"},{"name":"Innovates Health Solutions (AIHS) through an Alberta Cancer Foundation Grant; Senior Scientist"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Protein Science"],"published-print":{"date-parts":[[2015,8]]},"abstract":"<jats:title>Abstract<\/jats:title><jats:p>Water\u2010mediated hydrogen exchange (HX) processes involving the protein main chain are sensitive to structural dynamics and molecular interactions. Measuring deuterium uptake in amide bonds provides information on conformational states, structural transitions and binding events. Increasingly, deuterium levels are measured by mass spectrometry (MS) from proteolytically generated peptide fragments of large molecular systems. However, this bottom\u2010up method has limited spectral capacity and requires a burdensome manual validation exercise, both of which restrict analysis of protein systems to generally less than 150 kDa. In this study, we present a bottom\u2010up HX\u2010MS<jats:sup>2<\/jats:sup> method that improves peptide identification rates, localizes high\u2010quality HX data and simplifies validation. The method combines a new peptide scoring algorithm (WUF, weighted unique fragment) with data\u2010independent acquisition of peptide fragmentation data. Scoring incorporates the validation process and emphasizes identification accuracy. The HX\u2010MS<jats:sup>2<\/jats:sup> method is illustrated using data from a conformational analysis of microtubules treated with dimeric kinesin MCAK. When compared to a conventional Mascot\u2010driven HX\u2010MS method, HX\u2010MS<jats:sup>2<\/jats:sup> produces two\u2010fold higher \u03b1\/\u03b2\u2010tubulin sequence depth at a peptide utilization rate of 74%. A Mascot approach delivers a utilization rate of 44%. The WUF score can be constrained by false utilization rate (FUR) calculations to return utilization values exceeding 90% without serious data loss, indicating that automated validation should be possible. The HX\u2010MS<jats:sup>2<\/jats:sup> data confirm that N\u2010terminal MCAK domains anchor kinesin force generation in kinesin\u2010mediated depolymerization, while the C\u2010terminal tails regulate MCAK\u2010tubulin interactions.<\/jats:p>","DOI":"10.1002\/pro.2707","type":"journal-article","created":{"date-parts":[[2015,5,26]],"date-time":"2015-05-26T08:59:07Z","timestamp":1432630747000},"page":"1313-1324","source":"Crossref","is-referenced-by-count":9,"title":["<scp>HX\u2010MS<\/scp>2 for high performance conformational analysis of complex protein states"],"prefix":"10.1002","volume":"24","author":[{"given":"Kyle M.","family":"Burns","sequence":"first","affiliation":[{"name":"Department of Biochemistry and Molecular Biology and the Southern Alberta Cancer Research Institute University of Calgary Calgary Alberta Canada T2N 4N1"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Vladimir","family":"Sarpe","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Molecular Biology and the Southern Alberta Cancer Research Institute University of Calgary Calgary Alberta Canada T2N 4N1"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Mike","family":"Wagenbach","sequence":"additional","affiliation":[{"name":"Department of Physiology and Biophysics University of Washington School of Medicine Seattle Washington 98195\u20107290"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Linda","family":"Wordeman","sequence":"additional","affiliation":[{"name":"Department of Physiology and Biophysics University of Washington School of Medicine Seattle Washington 98195\u20107290"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"David C.","family":"Schriemer","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Molecular Biology and the Southern Alberta Cancer Research Institute University of Calgary Calgary Alberta Canada T2N 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