人低温离体灌注心脏心肌缺血损伤早期蛋白组学研究

目的分析离体心脏灌注早期心肌缺血损伤释放出的蛋白质组成,并探讨这些蛋白的生理学过程和功能。方法收集人离体心脏停跳后即刻、30 min和60 min后的4℃灌注液,样品首先用MARS柱去除高丰度蛋白,用10×103分子筛脱盐浓缩、溶液内酶解,得到肽段用mRP柱进一步脱盐,通过液相-芯片-离子阱质谱(HPLC-Chip-MS/MS)技术进行鉴定和生物信息学分析,建立离体心脏灌注早期心肌缺血释放蛋白全谱图,通过GO注释和EBI数据库对这些蛋白进行功能和定位分析以及BLAST比对。结果得到高可信蛋白176个(Score≥8,SPI≥70%);经GO注释分析,主要参与细胞、生物学调节、代谢等生理学过程,具有结合功能、催化活性以及酶调节活性等;通过EBI数据库对106×103未知蛋白和29×103未知蛋白进行BLAST比对发现前者是一种出胞蛋白,后者性质目前尚不明确。结论离体心脏灌注获得的176个高可信蛋白对进一步研究心肌缺血损伤早期生物学标记物具有重要的意义。106×103和29×103的2个未知蛋白可能是未发现的心肌早期缺血损伤蛋白标记物。

Proteomics in early myocardial ischemic injury of isolated human heart during hypothermia-perfusion

Objective To analyze the components of proteins released from early ischemic myocardial injury of isolated human heart during hypothermia perfusion and study their physiological processes and fun-ctions.Methods Perfusate at 4℃ was harvested from isolated human hearts instantly,30 and 60 min after arrest.Peptide fragments were obtained by removing abundant proteins from samples with MARS columns,desalinizing with a 10 kD molecular sieve,and digesting with enzyme in solution,desalinized again with mRP columns,identified by HPLC-Chip-MS/MS and analyzed.Spectra were established for all proteins released from early ischemic myocardial injury of isolated human heart during hypothermia perfusion.The functions and locations of these proteins were analyzed by GO and EBI,and compared with those of BLAST.Results A total of 176 high confidence proteins(score≥8,SPI≥70%) were obtained using the Agilent Spectrum Mill Home software.GO annotations showed that these proteins mainly participated in regulation of the physiological processes of cells,biology and metabolism,with blinding functions,catalytic activities and enzyme-modulating activities.The 106 kD agnoprotein was found to be an exocytosis protein by comparing 106 kD and 29 kD unknown proteins in BLAST and EBI database.Conclusion The 176 high-confidence proteins are of great significance for further study of biological markers for early myocardial ischemic injury,and the 106 and 29 kD agnoproteins may be two unknown proteins of early myocardial ischemic markers.