National Research Council of Italy

Institute of Biosciences and Bioresources

Protein/peptide sequencing

Figure 1: PPSQ protein/peptide sequencer

Figure 1: PPSQ protein/peptide sequencer

CloseFigure 1: PPSQ protein/peptide sequencer

Figure 1: PPSQ protein/peptide sequencer

Protein/Peptide Sequencing

The laboratory offers sensitive N-terminal protein/peptide sequencing using a recently installed Shimadzu PPSQ 33B sequencer (Figure 1) at IBBR-Napoli (Figure 2). The method is based on the Edman degradation reaction which labels the N-terminal amino acid residues within a polypeptide such that they can be cleaved from the chain in a stepwise manner (Figure 3). The benefits of using Edman degradation for protein sequencing are:

  • Guaranteed N-terminal sequence of proteins;
  • No sample pre-treatment required:
  • Differentiation of isobaric amino acids (leucine and isoleucine):
  • Sequencing of unknown proteins not registered in databases.

Preparing samples for sequencing

Figure 2: PPSQ 33 B at work at IBBR-CNR-Napoli

Figure 2: PPSQ 33 B at work at IBBR-CNR-Napoli

CloseFigure 2: PPSQ 33 B at work at IBBR-CNR-Napoli

Figure 2: PPSQ 33 B at work at IBBR-CNR-Napoli

Samples should contain a purified single protein or peptide and should be free of any reagents that interfere with the Edman degradation process, such as glycine, glycerol, sucrose, guanidine, primary amines, SDS, salts and amino acids. Samples in one of the following forms should be for the analysis:

  1. lyophilized
  2. in solution
  3. immobilized on PVDF membrane (a Coomassie stainable amount)

Figure 3: Schematics of the workflow used by the sequencer.

Figure 3: Schematics of the workflow used by the sequencer.

CloseFigure 3: Schematics of the workflow used by the sequencer.

Figure 3: Schematics of the workflow used by the sequencer.

In the case of cysteine residue present in the N-terminal portion, N-terminal blockage, post-translational modifications and a more detailed guide for sample preparation guide, please ask directly in laboratory (+39-0816132585/613) or email to immacolata.fiume@ibbr.cnr.it.

 

by: Gabriele Bucci - Last Updated: Aug 14, 2016 (17:05)