Alayi Tchilabalo D, Tawalbeh Shefa M, Ogundele Michael, Smith Holly R, Samsel Alison M, Barbieri Marissa L, Hathout Yetrib
Department of Pharmaceutical Science, School of Pharmacy and Pharmaceutical Sciences, Binghamton University-SUNY, Johnson City, New York 13790, United States.
Department of Biomedical Engineering, Binghamton University-SUNY, 4400 Vestal Pkwy E, Binghamton, New York 13902, United States.
ACS Omega. 2020 Oct 6;5(41):26504-26517. doi: 10.1021/acsomega.0c03206. eCollection 2020 Oct 20.
Blood-accessible molecular biomarkers are becoming highly attractive tools to assess disease progression and response to therapies in Duchenne muscular dystrophy (DMD) especially in very young patients for whom other outcome measures remain subjective and challenging. 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 ( = 9) and age-matched healthy controls ( = 9) identified 38 elevated and 50 decreased serum proteins (adjusted < 0.05, FDR <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-glucose-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 < 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.
血液中可检测到的分子生物标志物正成为评估杜氏肌营养不良症（DMD）疾病进展和治疗反应的极具吸引力的工具，尤其是对于非常年幼的患者，其他结局指标仍然主观且具有挑战性。在本研究中，我们使用串联质量标签（TMT）策略，结合血清中丰富蛋白质的去除和高pH反相肽分级分离，标准化了一种高度特异且可重复的多重质谱方法。对9名4岁DMD男孩和9名年龄匹配的健康对照进行差异蛋白质组分析，结果显示，与健康对照组相比，DMD组中有38种血清蛋白升高，50种血清蛋白降低（校正P<0.05，错误发现率<0.05）。正如预期的那样，我们证实了先前报道的生物标志物，但也发现了新的生物标志物。这些新生物标志物包括与肌肉损伤相关的新生物标志物，如隐钙素、类平滑肌蛋白1、丝切蛋白-1和网蛋白；其他肌肉特异性酶，如尿苷三磷酸-葡萄糖-1-磷酸尿苷酰转移酶、天冬氨酸转氨酶、丙酮酸激酶PKM、乳铁转铁蛋白、组织α-L-岩藻糖苷酶、泛硫乙胺酶和纤维胶凝蛋白-1；以及一些与促炎和细胞黏附相关的生物标志物，如白细胞涎酸蛋白、巨噬细胞受体MARCO、玻连蛋白、半乳糖凝集素-3结合蛋白和前体分泌蛋白原相关神经肽。包括血清去除、样品处理和质谱分析在内的工作流程随着时间的推移具有可重复性和稳定性，变异系数<20%。此外，与其他基于多重亲和的方法相比，该方法在特异性方面表现更优。这些发现证明了基于TMT的质谱方法在检测和识别症状前年轻DMD患者血清生物标志物方面的特异性和可靠性。
