心血管疾病(CVDs)是全球范围内的主要死亡原因,CVD的患病率随着年龄的增长而明显增加。由于需求旺盛,心脏对线粒体功能障碍高度敏感。心脏线粒体蛋白质组的复杂性阻碍了针对CVD中线粒体功能障碍的有效策略的发展。哺乳动物线粒体由超过1000种蛋白质组成,其中大多数可以进行翻译后蛋白质修饰(PTM)。自上而下的蛋白质组学是表征和定量所有蛋白质序列变异和PTM的强大技术。然而,使用该技术研究与年龄相关的线粒体蛋白质形态变化仍存在知识空白.在这项研究中,我们使用自上而下的蛋白质组学鉴定了年轻和老年心脏中完整的线粒体蛋白质形式,并确定了心脏衰老过程中蛋白质丰度和PTM的变化.方法:从年轻(4个月大)和老年(24-25个月大)小鼠的心脏中分离完整的线粒体。线粒体被裂解了,和线粒体裂解物进行变性,reduction,和烷基化。对于定量自上而下的分析,在两个条件下,总共有12次来自3次生物重复,每个样品的技术副本。对收集的自上而下的数据集进行去卷积和量化,然后鉴定了蛋白形式。结果:从总共12次LC-MS/MS运行中,我们在不同的亚线粒体区室中鉴定出134种独特的线粒体蛋白(OMM,IMS,IMM,矩阵)。鉴定了不同质量范围的823种独特的蛋白形式。与年轻小鼠的心脏线粒体相比,在老年小鼠的心脏线粒体中,7种蛋白形式的丰度增加,13种蛋白形式的丰度降低。我们的分析还检测到线粒体蛋白形式的PTM,包括N端乙酰化,赖氨酸琥珀酰化,赖氨酸乙酰化,氧化,和磷酸化。结论:通过使用线粒体分级分离的线粒体蛋白质富集与使用超高压液相色谱(UPLC)-MS和无标记定量的自上而下的定量分析相结合,我们成功地鉴定并定量了复杂线粒体蛋白质组中完整的蛋白质形式.使用这种方法,我们检测到心脏线粒体蛋白质的丰度和PTM的年龄相关变化.
Cardiovascular diseases (CVDs) are the leading cause of death worldwide, and the prevalence of CVDs increases markedly with age. Due to the high energetic demand, the heart is highly sensitive to mitochondrial dysfunction. The complexity of the cardiac mitochondrial proteome hinders the development of effective strategies that target mitochondrial dysfunction in CVDs. Mammalian mitochondria are composed of over 1000 proteins, most of which can undergo post-translational protein modifications (PTMs). Top-down proteomics is a powerful technique for characterizing and quantifying all protein sequence variations and PTMs. However, there are still knowledge gaps in the study of age-related mitochondrial proteoform changes using this technique. In this study, we used top-down proteomics to identify intact mitochondrial proteoforms in young and old hearts and determined changes in protein abundance and PTMs in cardiac aging.
METHODS: Intact mitochondria were isolated from the hearts of young (4-month-old) and old (24-25-month-old) mice. The mitochondria were lysed, and mitochondrial lysates were subjected to denaturation, reduction, and alkylation. For quantitative top-down analysis, there were 12 runs in total arising from 3 biological replicates in two conditions, with technical duplicates for each sample. The collected top-down datasets were deconvoluted and quantified, and then the proteoforms were identified.
RESULTS: From a total of 12 LC-MS/MS runs, we identified 134 unique mitochondrial proteins in the different sub-mitochondrial compartments (OMM, IMS, IMM, matrix). 823 unique proteoforms in different mass ranges were identified. Compared to cardiac mitochondria of young mice, 7 proteoforms exhibited increased abundance and 13 proteoforms exhibited decreased abundance in cardiac mitochondria of old mice. Our analysis also detected PTMs of mitochondrial proteoforms, including N-terminal acetylation, lysine succinylation, lysine acetylation, oxidation, and phosphorylation.
CONCLUSIONS: By combining mitochondrial protein enrichment using mitochondrial fractionation with quantitative top-down analysis using ultrahigh-pressure liquid chromatography (UPLC)-MS and label-free quantitation, we successfully identified and quantified intact proteoforms in the complex mitochondrial proteome. Using this approach, we detected age-related changes in abundance and PTMs of mitochondrial proteoforms in the heart.