N-Terminal Amino Acid Sequence Determination of Proteins by N-Terminal
Dimethyl Labeling: Pitfalls and Advantages When Compared with Edman Degradation
Sequence Analysis |
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| Authors: | Elizabeth Chang Sergei Pourmal Chun Zhou Rupesh Kumar Marianna Teplova Nikola P. Pavletich Kenneth J. Marians Hediye Erdjument-Bromage |
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| Affiliation: | 1Microchemistry and Proteomics Core Laboratory, 2Structural Biology and 3Molecular Biology Program, Memorial Sloan Kettering Cancer Center, New York, New York, USA |
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| Abstract: | In recent history, alternative approaches to Edman sequencing have been investigated,and to this end, the Association of Biomolecular Resource Facilities (ABRF) ProteinSequencing Research Group (PSRG) initiated studies in 2014 and 2015, looking intobottom-up and top-down N-terminal (Nt) dimethyl derivatization of standard quantitiesof intact proteins with the aim to determine Nt sequence information. We haveexpanded this initiative and used low picomole amounts of myoglobin to determine theefficiency of Nt-dimethylation. Application of this approach on protein domains,generated by limited proteolysis of overexpressed proteins, confirms that it is auniversal labeling technique and is very sensitive when compared with Edmansequencing. Finally, we compared Edman sequencing and Nt-dimethylation of the samepolypeptide fragments; results confirm that there is agreement in the identity of theNt amino acid sequence between these 2 methods. |
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| Keywords: | Nt protein dimethylation Nt sequencing bottom-up MS in-gel digestion |
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