Protocol

Two-Dimensional Electrophoresis Protocols

Volume 519 of the series Methods in Molecular Biology pp 495-506

Date:

de Novo Sequence Analysis of N-Terminal Sulfonated Peptides After in-Gel Guanidination

  • Kjell SergeantAffiliated withLaboratory of Protein Biochemistry and Protein Engineering, Department of Biochemistry, Physiology and Microbiology, Ghent University
  • , Jozef Van BeeumenAffiliated withLaboratory of Protein Biochemistry and Protein Engineering, Department of Biochemistry, Physiology and Microbiology, Ghent University
  • , Bart SamynAffiliated withLaboratory of Protein Biochemistry and Protein Engineering, Department of Biochemistry, Physiology and Microbiology, Ghent University Email author 

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Summary

In this protocol, we describe an approach in which two-dimensional electrophoresis (2DE)-separated proteins are guanidinated in-gel prior to enzymatic cleavage. In contrast to previously described techniques, this procedure allows the extracted tryptic peptides to be N-terminally sulfonated without any further sample purification. The protocol was applied on a proteomic study of 2DE-separated proteins from Halorhodospira halophila, an extremophilic eubacterium with an unsequenced genome at the moment of analysis.

Key words

Chemically assisted fragmentation Guanidination Sulfonation De novo sequence analysis