Elizabeth Chang, S. Pourmal, Chun Zhou, Rupesh Kumar, M. Teplova, N. Pavletich, K. Marians, H. Erdjument-Bromage
{"title":"n端二甲基标记法测定蛋白质n端氨基酸序列:与Edman降解序列分析相比的缺陷与优势","authors":"Elizabeth Chang, S. Pourmal, Chun Zhou, Rupesh Kumar, M. Teplova, N. Pavletich, K. Marians, H. Erdjument-Bromage","doi":"10.7171/jbt.16-2702-002","DOIUrl":null,"url":null,"abstract":"In recent history, alternative approaches to Edman sequencing have been investigated, and to this end, the Association of Biomolecular Resource Facilities (ABRF) Protein Sequencing Research Group (PSRG) initiated studies in 2014 and 2015, looking into bottom-up and top-down N-terminal (Nt) dimethyl derivatization of standard quantities of intact proteins with the aim to determine Nt sequence information. We have expanded this initiative and used low picomole amounts of myoglobin to determine the efficiency of Nt-dimethylation. Application of this approach on protein domains, generated by limited proteolysis of overexpressed proteins, confirms that it is a universal labeling technique and is very sensitive when compared with Edman sequencing. Finally, we compared Edman sequencing and Nt-dimethylation of the same polypeptide fragments; results confirm that there is agreement in the identity of the Nt amino acid sequence between these 2 methods.","PeriodicalId":94326,"journal":{"name":"Journal of biomolecular techniques : JBT","volume":"8 1","pages":"61-74"},"PeriodicalIF":0.0000,"publicationDate":"2016-03-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"10","resultStr":"{\"title\":\"N-Terminal Amino Acid Sequence Determination of Proteins by N-Terminal Dimethyl Labeling: Pitfalls and Advantages When Compared with Edman Degradation Sequence Analysis.\",\"authors\":\"Elizabeth Chang, S. Pourmal, Chun Zhou, Rupesh Kumar, M. Teplova, N. Pavletich, K. Marians, H. Erdjument-Bromage\",\"doi\":\"10.7171/jbt.16-2702-002\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"In recent history, alternative approaches to Edman sequencing have been investigated, and to this end, the Association of Biomolecular Resource Facilities (ABRF) Protein Sequencing Research Group (PSRG) initiated studies in 2014 and 2015, looking into bottom-up and top-down N-terminal (Nt) dimethyl derivatization of standard quantities of intact proteins with the aim to determine Nt sequence information. We have expanded this initiative and used low picomole amounts of myoglobin to determine the efficiency of Nt-dimethylation. Application of this approach on protein domains, generated by limited proteolysis of overexpressed proteins, confirms that it is a universal labeling technique and is very sensitive when compared with Edman sequencing. Finally, we compared Edman sequencing and Nt-dimethylation of the same polypeptide fragments; results confirm that there is agreement in the identity of the Nt amino acid sequence between these 2 methods.\",\"PeriodicalId\":94326,\"journal\":{\"name\":\"Journal of biomolecular techniques : JBT\",\"volume\":\"8 1\",\"pages\":\"61-74\"},\"PeriodicalIF\":0.0000,\"publicationDate\":\"2016-03-07\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"10\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Journal of biomolecular techniques : JBT\",\"FirstCategoryId\":\"1085\",\"ListUrlMain\":\"https://doi.org/10.7171/jbt.16-2702-002\",\"RegionNum\":0,\"RegionCategory\":null,\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"\",\"JCRName\":\"\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Journal of biomolecular techniques : JBT","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.7171/jbt.16-2702-002","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"","JCRName":"","Score":null,"Total":0}
N-Terminal Amino Acid Sequence Determination of Proteins by N-Terminal Dimethyl Labeling: Pitfalls and Advantages When Compared with Edman Degradation Sequence Analysis.
In recent history, alternative approaches to Edman sequencing have been investigated, and to this end, the Association of Biomolecular Resource Facilities (ABRF) Protein Sequencing Research Group (PSRG) initiated studies in 2014 and 2015, looking into bottom-up and top-down N-terminal (Nt) dimethyl derivatization of standard quantities of intact proteins with the aim to determine Nt sequence information. We have expanded this initiative and used low picomole amounts of myoglobin to determine the efficiency of Nt-dimethylation. Application of this approach on protein domains, generated by limited proteolysis of overexpressed proteins, confirms that it is a universal labeling technique and is very sensitive when compared with Edman sequencing. Finally, we compared Edman sequencing and Nt-dimethylation of the same polypeptide fragments; results confirm that there is agreement in the identity of the Nt amino acid sequence between these 2 methods.