Becker Edgar, Francino Antonio, Pich Andreas, Perrot Andreas, Kraft Theresia, Radocaj Ante
Institute for Molecular and Cell Physiology, Hannover Medical School, Hannover, Germany.
Cardiology Department, Hospital Clinic/IDIBAPS, University of Barcelona, Barcelona, Spain.
Front Cardiovasc Med. 2022 Feb 21;9:816330. doi: 10.3389/fcvm.2022.816330. eCollection 2022.
In genetic diseases like hypertrophic cardiomyopathy, reliable quantification of the expression level of mutant protein can play an important role in disease research, diagnosis, treatment and prognosis. For heterozygous β-myosin heavy chain (β-MyHC) mutations it has been shown that disease severity is related to the fraction of mutant protein in the myocardium. Yet, heart tissue from patients with genetically characterized diseases is scarce. Here we asked, if even in the case of small endomyocardial biopsies, single quantifications produce reliable results. Myocardial samples were taken from four different regions of an explanted heart of a patient with hypertrophic cardiomyopathy carrying point mutation p.Gly716Arg in β-MyHC. From both, large samples (15 mg) and small, endomyocardial biopsy-sized samples (≤ 1 mg) myosin was extracted and enzymatically digested to yield a specific peptide of interest that allowed to distinguish mutant and wild-type β-MyHC. Absolute quantification by mass spectrometry (AQUA) of the peptide of interest was performed repeatedly for both sample sizes to determine the fraction of mutant β-MyHC. Fractions of mutant β-MyHC (32% on average) showed only small differences between the four cardiac regions and for large and small samples. The standard deviations were smaller than five percentage points for all cardiac regions. The two quantification methods (large and small sample size) produce results with comparable accuracy and precision. Consequently, with our method even small endomyocardial biopsies allow reliable protein quantification for potential diagnostic purposes.
在肥厚型心肌病等遗传性疾病中，对突变蛋白表达水平进行可靠定量在疾病研究、诊断、治疗和预后方面可发挥重要作用。对于杂合β - 肌球蛋白重链（β - MyHC）突变，研究表明疾病严重程度与心肌中突变蛋白的比例有关。然而，具有明确遗传特征疾病患者的心脏组织稀缺。在此我们探讨，即使是对于小的心内膜活检样本，单次定量是否能产生可靠结果。从一名携带β - MyHC基因p.Gly716Arg点突变的肥厚型心肌病患者的离体心脏的四个不同区域采集心肌样本。从大样本（15毫克）和小的心内膜活检大小的样本（≤1毫克）中提取肌球蛋白并进行酶消化，以产生一种特定的目标肽，该肽可用于区分突变型和野生型β - MyHC。对两种样本大小的目标肽均反复进行质谱绝对定量（AQUA），以确定突变型β - MyHC的比例。突变型β - MyHC的比例（平均32%）在四个心脏区域以及大样本和小样本之间仅显示出微小差异。所有心脏区域的标准差均小于5个百分点。两种定量方法（大样本和小样本）产生的结果具有相当的准确性和精密度。因此，使用我们的方法，即使是小的心内膜活检样本也能进行可靠的蛋白质定量，用于潜在的诊断目的。
