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J Biomol Tech. 2016 Jul;27(2):61-74. doi: 10.7171/jbt.16-2702-002. Epub 2016 Mar 7.

N-Terminal Amino Acid Sequence Determination of Proteins by N-Terminal Dimethyl Labeling: Pitfalls and Advantages When Compared with Edman Degradation Sequence Analysis.

Author information

1
1 Microchemistry and Proteomics Core Laboratory, 2 Structural Biology and 3 Molecular Biology Program, Memorial Sloan Kettering Cancer Center, New York, New York, USA.

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.

KEYWORDS:

Nt protein dimethylation; Nt sequencing; bottom-up MS; in-gel digestion

PMID:
27006647
PMCID:
PMC4802820
DOI:
10.7171/jbt.16-2702-002
[Indexed for MEDLINE]
Free PMC Article

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