Fernández-Guerra Paula, Birkler Rune I D, Merinero Begoña, Ugarte Magdalena, Gregersen Niels, Rodríguez-Pombo Pilar, Bross Peter, Palmfeldt Johan
Research Unit for Molecular Medicine, Department of Clinical Medicine, Aarhus University and Aarhus University Hospital Aarhus, Denmark.
Centro de Diagnóstico de Enfermedades Moleculares (CEDEM), Centro de Investigación en Red de Enfermedades Raras (CIBERER), IDIPAZ, Universidad Autónoma Madrid Madrid, Spain.
Mol Genet Genomic Med. 2014 Sep;2(5):383-92. doi: 10.1002/mgg3.88. Epub 2014 Jun 4.
Selected reaction monitoring (SRM) mass spectrometry can quantitatively measure proteins by specific targeting of peptide sequences, and allows the determination of multiple proteins in one single analysis. Here, we show the feasibility of simultaneous measurements of multiple proteins in mitochondria-enriched samples from cultured fibroblasts from healthy individuals and patients with mutations in branched-chain α-ketoacid dehydrogenase (BCKDH) complex. BCKDH is a mitochondrial multienzyme complex and its defective activity causes maple syrup urine disease (MSUD), a rare but severe inherited metabolic disorder. Four different genes encode the catalytic subunits of BCKDH: E1α (BCKDHA), E1β (BCKDHB), E2 (DBT), and E3 (DLD). All four proteins were successfully quantified in healthy individuals. However, the E1α and E1β proteins were not detected in patients carrying mutations in one of those genes, whereas mRNA levels were almost unaltered, indicating instability of E1α and E1β monomers. Using SRM we elucidated the protein effects of mutations generating premature termination codons or misfolded proteins. SRM is a complement to transcript level measurements and a valuable tool to shed light on molecular mechanisms and on effects of pharmacological therapies at protein level. SRM is particularly effective for inherited disorders caused by multiple proteins such as defects in multienzyme complexes.
选择反应监测（SRM）质谱分析法能够通过对肽序列进行特异性靶向来定量测量蛋白质，并允许在一次分析中测定多种蛋白质。在此，我们展示了在来自健康个体以及患有支链α-酮酸脱氢酶（BCKDH）复合物突变的患者的培养成纤维细胞的富含线粒体的样本中同时测量多种蛋白质的可行性。BCKDH是一种线粒体多酶复合物，其活性缺陷会导致枫糖尿症（MSUD），这是一种罕见但严重的遗传性代谢紊乱疾病。四种不同的基因编码BCKDH的催化亚基：E1α（BCKDHA）、E1β（BCKDHB）、E2（DBT）和E3（DLD）。在健康个体中成功对所有这四种蛋白质进行了定量。然而，在携带这些基因之一发生突变的患者中未检测到E1α和E1β蛋白，而mRNA水平几乎未改变，这表明E1α和E1β单体不稳定。我们使用SRM阐明了产生过早终止密码子或错误折叠蛋白的突变的蛋白质效应。SRM是转录水平测量的一种补充，并且是一种在蛋白质水平上阐明分子机制和药物治疗效果的有价值工具。SRM对于由多种蛋白质引起的遗传性疾病（如多酶复合物缺陷）特别有效。
