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N-Terminal protein sequencing by nicotinic acid derivatization and MS analysis
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DOI：10.1007/s00216-026-06727-4.png)
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Abstract
En 中文
Most MS-based workflows for N-terminal protein sequence analysis rely on the modification of primary amines at the protein N-terminus and lysine residues, in many cases through dimethylation. However, incomplete dimethyl labeling and undesired side reactions have been reported previously and were also observed in our experiments. In this study, we evaluated alternative methods for N-terminal protein derivatization. For reductive dimethylation, the highly toxic reducing agent sodium cyanoborohydride was successfully replaced by pyridine borane. In addition, several single-reagent labeling methods were applied to model proteins and compared to the dimethylation workflow. Criteria for the selection of suitable reagents were completeness and selectivity of the protein derivatization reaction, the detection sensitivity for modified peptides by mass spectrometry, and the absence of undesirable side reactions. Comparative analyses demonstrated that the chemical derivatization of free amines by nicotinic acid N-hydroxysuccinimide ester is a particularly promising approach for N-terminal protein sequencing. Protocols for the nicotinoylation of proteins in solution or in gel using this reagent were optimized and successfully applied to various test proteins.
Keywords:
N-Terminomics
N-Terminal
Protein sequencing
Dimethylation
Nicotinic acid
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