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In this study, we have standardized a highly specific and reproducible multiplexing mass spectrometry method using the tandem mass tag (TMT) strategy in combination with depletion of abundant proteins from serum and high-pH reversed-phase peptide fractionation. Differential proteome profiling of 4 year-old DMD boys (n = 9) and age-matched healthy controls (n = 9) identified 38 elevated and 50 decreased serum proteins (adjusted P &amp;lt; 0.05, FDR &amp;lt;0.05) in the DMD group relative to the healthy control group. As expected, we confirmed previously reported biomarkers but also identified novel biomarkers. These included novel muscle injury-associated biomarkers such as telethonin, smoothelin-like protein 1, cofilin-1, and plectin, additional muscle-specific enzymes such as UTP\u2013glucose-1-phosphate uridylyltransferase, aspartate aminotransferase, pyruvate kinase PKM, lactotransferrin, tissue alpha-l-fucosidase, pantetheinase, and ficolin-1, and some pro-inflammatory and cell adhesion-associated biomarkers such as leukosialin, macrophage receptor MARCO, vitronectin, galectin-3-binding protein, and ProSAAS. The workflow including serum depletion, sample processing, and mass spectrometry analysis was found to be reproducible and stable over time with CV &amp;lt; 20%. Furthermore, the method was found to be superior in terms of specificity compared to other multiplexing affinity-based methods. These findings demonstrate the specificity and reliability of TMT-based mass spectrometry methods in detection and identification of serum biomarkers in presymptomatic young DMD patients.<\/jats:p>","DOI":"10.1021\/acsomega.0c03206","type":"journal-article","created":{"date-parts":[[2020,10,6]],"date-time":"2020-10-06T12:34:52Z","timestamp":1601987692000},"page":"26504-26517","source":"Crossref","is-referenced-by-count":19,"title":["Tandem Mass Tag-Based Serum Proteome Profiling for\nBiomarker Discovery in Young Duchenne Muscular Dystrophy Boys"],"prefix":"10.1021","volume":"5","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-7291-531X","authenticated-orcid":true,"given":"Tchilabalo D.","family":"Alayi","sequence":"first","affiliation":[{"name":"Binghamton University\u2212SUNY , , , ,","place":["Johnson City, New York, United States, 13790"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shefa M.","family":"Tawalbeh","sequence":"additional","affiliation":[{"name":"Binghamton University\u2212SUNY , , , ,","place":["Johnson City, New York, United States, 13790"]},{"name":"Binghamton University\u2212SUNY , , 4400 Vestal Pkwy E , , ,","place":["Binghamton, New York, United States, 13902"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Michael","family":"Ogundele","sequence":"additional","affiliation":[{"name":"Binghamton University\u2212SUNY , , , ,","place":["Johnson City, New York, United States, 13790"]},{"name":"Binghamton University\u2212SUNY , , 4400 Vestal Pkwy E , , ,","place":["Binghamton, New York, United States, 13902"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Holly R.","family":"Smith","sequence":"additional","affiliation":[{"name":"Binghamton University\u2212SUNY , , , ,","place":["Johnson City, New York, United States, 13790"]},{"name":"Binghamton University\u2212SUNY , , 4400 Vestal Pkwy E , , ,","place":["Binghamton, New York, United States, 13902"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alison M.","family":"Samsel","sequence":"additional","affiliation":[{"name":"Binghamton University\u2212SUNY , , , ,","place":["Johnson City, New York, United States, 13790"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Marissa L.","family":"Barbieri","sequence":"additional","affiliation":[{"name":"Binghamton University\u2212SUNY , , , ,","place":["Johnson City, New York, United States, 13790"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yetrib","family":"Hathout","sequence":"additional","affiliation":[{"name":"Binghamton University\u2212SUNY , , , ,","place":["Johnson City, New York, United States, 13790"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2020,10,6]]},"reference":[{"key":"2026090206453642300_cit1","doi-asserted-by":"publisher","first-page":"304","DOI":"10.1002\/ana.23528","article-title":"Evidence-Based Path\nto Newborn Screening for Duchenne\nMuscular Dystrophy","volume":"71","author":"Mendell","year":"2012","journal-title":"Annu. Neurol."},{"key":"2026090206453642300_cit2","doi-asserted-by":"publisher","first-page":"281","DOI":"10.1146\/annurev-genom-090314-025003","article-title":"The Pathogenesis and Therapy of Muscular Dystrophies","volume":"16","author":"Guiraud","year":"2015","journal-title":"Annu. Rev. Genomics Hum. Genet."},{"key":"2026090206453642300_cit3","doi-asserted-by":"publisher","first-page":"1275","DOI":"10.1177\/0883073814558120","article-title":"Corticosteroid\nTreatments in Males With Duchenne Muscular Dystrophy: Treatment Duration\nand Time to Loss of Ambulation","volume":"30","author":"Kim","year":"2015","journal-title":"J. Child Neurol."},{"key":"2026090206453642300_cit4","doi-asserted-by":"publisher","first-page":"492","DOI":"10.1002\/phar.1909","article-title":"Advances in the\nTreatment of Duchenne\nMuscular Dystrophy: New and Emerging Pharmacotherapies","volume":"37","author":"Reinig","year":"2017","journal-title":"Pharmacotherapy"},{"key":"2026090206453642300_cit5","doi-asserted-by":"publisher","first-page":"1","DOI":"10.3390\/jpm9010001","article-title":"Restoring Dystrophin\nExpression in Duchenne Muscular Dystrophy: Current\nStatus of Therapeutic Approaches","volume":"9","author":"Shimizu-Motohashi","year":"2019","journal-title":"J. Pers. Med."},{"key":"2026090206453642300_cit6","doi-asserted-by":"publisher","first-page":"343","DOI":"10.1002\/mus.23902","article-title":"The 6-Minute\nWalk Test and Other Endpoints in Duchenne Muscular Dystrophy: Longitudinal\nNatural History Observations over 48 Weeks from a Multicenter Study","volume":"48","author":"McDonald","year":"2013","journal-title":"Muscle Nerve"},{"key":"2026090206453642300_cit7","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0160195","article-title":"Revised North Star\nAmbulatory Assessment for Young Boys with Duchenne Muscular Dystrophy","volume":"11","author":"Mercuri","year":"2016","journal-title":"PLoS One"},{"key":"2026090206453642300_cit8","doi-asserted-by":"publisher","first-page":"9","DOI":"10.1186\/s12014-016-9109-x","article-title":"Clinical Utility of Serum Biomarkers in Duchenne Muscular\nDystrophy","volume":"13","author":"Hathout","year":"2016","journal-title":"Clin. 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Neuromuscular Dis."},{"key":"2026090206453642300_cit32","doi-asserted-by":"publisher","first-page":"2482","DOI":"10.1161\/CIRCULATIONAHA.118.036823","article-title":"In Aptamers They Trust: The Caveats\nof the SOMAscan Biomarker Discovery\nPlatform from SomaLogic","volume":"138","author":"Joshi","year":"2018","journal-title":"Circulation"},{"key":"2026090206453642300_cit33","doi-asserted-by":"publisher","first-page":"36","DOI":"10.1093\/clinchem\/37.1.36","article-title":"Carbonic Anhydrase\nIII in Serum in Muscular Dystrophy and Other Neurological\nDisorders: Relationship with Creatine Kinase","volume":"37","author":"Ohta","year":"1991","journal-title":"Clin. Chem."},{"key":"2026090206453642300_cit34","doi-asserted-by":"publisher","first-page":"785","DOI":"10.1016\/j.molimm.2003.10.002","article-title":"Carboxypeptidase N: A Pleiotropic\nRegulator of Inflammation","volume":"40","author":"Matthews","year":"2004","journal-title":"Mol. 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