Rationale: Cell therapy for myocardial infarction is promising but largely unsuccessful in part due to a lack of mechanistic understanding. Techniques enabling identification of stem cell-specific proteomes in situ in the injured heart may shed light on how the administered cells respond to the injured microenvironment and exert reparative effects./nObjective: To identify the proteomes of the transplanted mesenchymal stem cells (MSCs) in the infarcted myocardium, we sought to target a mutant methionyl-tRNA synthetase (MetRS^L274G) in MSCs, which charges azidonorleucine (ANL), a methionine analogue and non-canonical amino acid, to tRNA and subsequently to nascent proteins, permitting isolation of ANL-labeled MSC proteomes from ischemic hearts by ANL-alkyne based click reaction./nMethods and Results: Murine MSCs were transduced with lentivirus MetRS^L274G and supplemented with ANL; the ANL-tagged nascent proteins were visualized by bio-orthogonal non-canonical amino-acid tagging, spanning all molecular weights and by fluorescent non-canonical amino-acid tagging, displaying strong fluorescent signal. Then, the MetRS^L274G-transduced MSCs were administered to the infarcted or Sham heart in mice receiving ANL treatment. The MSC proteomes were isolated from the left ventricular protein lysates by click reaction at days 1, 3, and 7 after cell administration, identified by LC/MS. Among all identified proteins (in Sham and MI hearts, three time-points each), 648 were shared by all 6 groups, accounting for 82±5% of total proteins in each group, and enriched under mitochondrion, extracellular exosomes, oxidation-reduction process and poly(A) RNA binding. Notably, 26, 110 and 65 proteins were significantly up-regulated and 11, 28 and 19 proteins were down-regulated in the infarcted vs. Sham heart at the three time-points, respectively; these proteins are pronounced in the GO terms of extracellular matrix organization, response to stress and regulation of apoptotic process and in the KEGG pathways of complements and coagulation cascades, apoptosis, and regulators of actin cytoskeleton./nConclusions: MetRS^L274G expression allows successful identification of MSC-specific nascent proteins in the infarcted hearts, which reflect the functional states, adaptive response, and reparative effects of MSCs that may be leveraged to improve cardiac repair.

中文翻译：

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缺血心脏原位间充质干细胞蛋白质组学分析

理由：用于心肌梗塞的细胞疗法是有前途的，但在很大程度上由于缺乏对机械学的了解而未能成功。能够在受伤的心脏中原位鉴定干细胞特异性蛋白质组的技术可以揭示所施用的细胞如何对受损的微环境做出反应并发挥修复作用。/n目的：鉴定移植的间充质干细胞（MSCs）的蛋白质组。在梗死的心肌中，我们试图靶向突变的甲硫氨酰-tRNA合成酶（MetRS ^L274G）中的MSCs中，将azidonorleucine（ANL）（一种蛋氨酸类似物和非规范性氨基酸）带入tRNA并随后带入新生蛋白质中，从而可以通过基于ANL-炔烃的点击反应从缺血性心脏中分离出ANL标记的MSC蛋白质组。方法与结果：用慢病毒MetRS ^L274G转导小鼠骨髓间充^质干^细胞并补充ANL。通过生物正交的非规范氨基酸标签（覆盖所有分子量）和荧光非规范氨基酸标签（显示强荧光信号），可以可视化ANL标记的新生蛋白质。然后，MetRS ^L274G在接受ANL治疗的小鼠中，将经转导的MSCs给予梗塞或Sham心脏。通过LC / MS鉴定，在细胞给药后第1、3和7天通过点击反应从左心室蛋白裂解物中分离出MSC蛋白组。在所有鉴定出的蛋白质中（在Sham和MI心脏中，每个三个时间点），所有6组共有648个，占每组总蛋白质的82±5％，并在线粒体，细胞外囊泡，氧化还原下​​富集过程和poly（A）RNA结合。值得注意的是，在三个时间点，梗死组与假手术组的心脏中分别有26、110和65个蛋白上调，而11、28和19个蛋白下调。这些蛋白质在细胞外基质组织的GO术语中是明显的，结论： MetRS ^L274G的表达可以成功鉴定梗死心脏中MSC的新生蛋白质，这些蛋白质反映了MSC的功能状态，适应性反应和修复作用，可用于改善心脏修复。