Protein Sequencing and Analysis

TaKaRa Bio

1) Protein Fragmentation

Acylamino-acid-releasing enzyme

• Efficiently liberate the N-terminal acetylamino acid from N-acetylated peptides of up to about 30 residues in length
• It does not act on intact N-acetylated proteins
• Fragmentation of proteins with residue specific proteases followed by the release of the N-acetylamino acid from the isolated peptides is required to facilitate N-terminal sequence analysis of proteins.

 

Arginylendopeptidase: Protein Digestion

• Cleaves peptide bonds at the carboxyl side of arginine residues located on proteins and peptides
• Fragmentation of proteins and peptides prior to structural analysis
• Has been treated with TLCK and TPCK to remove trace trypsin-like and chymotrypsin-like protease activities
• Supplied with a 5X Reaction Buffer [250 mM sodium phosphate buffer (pH 8.0 )]

 

Asparaginylendopeptidase: Protein Analysis

• Peptide bond cleavage
• Protein/peptide fragmentation prior to structural analysis
• Specifically cleaves peptide bonds on the carboxyl side of asparagine residues including –Asn-Pro– bonds

 

Endoproteinase Asp-N: Protein Fragmentation for Sequencing

• Fragmentation of proteins and peptides required for primary structure analysis
• Endoproteinase Asp-N is a metalloprotease that hydrolyzes peptide bonds on the amino terminus of aspartic and cysteic acid (oxidized cysteine)
• The enzyme is supplied with a 250 mM sodium phosphate buffer (pH 8.0)

 

Pfu Protease S: Protein Digestion for Structural Analysis

• Endo-type serine protease with broad specificity for native and denatured proteins
• Cleavage occurs mainly on the carboxy terminus of peptide bonds on hydrophobic amino acid residues

 

2) N-terminal Deblocking and Analysis

Based on Edman Degradation and Protein Sequencing. Edman degradation is a commonly used method for protein sequencing via N-terminal amino acid cleavage.

 

Pfu Aminopeptidase I

• The Pfu Aminopeptidase I is a thermostable exo-type aminopeptidase isolated from Pyrococcus furiosus.
• It is expressed as a recombinant protein and liberates N-terminal amino acids from proteins and peptides
• Liberates the N-terminal amino acids up to X-Pro from proteins and peptides
• It does not hydrolyze peptide bonds at the α-amino group of proline (X-Pro)
• Enzyme activity is significantly increased in the presence of Co²⁺

 

Pfu Methionine Aminopeptidase

• Thermostable methionine aminopeptidase isolated from Pyrococcus furiosus
• Expressed as a recombinant protein
• Specifically liberates the N-terminal methionine residue from proteins and peptides during Edman degradation, enabling protein sequencing

 

Pfu N-acetyl Deblocking Aminopeptidase (Ac-DAP)

• Unique exo-type aminopeptidase that liberates blocking groups (formyl, acetyl, and myristyl) from proteins and peptides
• Releasing amino acids until it reaches the first X-Pro bond
• Used to determine short stretches of amino acid sequence in blocked proteins and peptides via mass spectrometry
• Since its amino terminus is acetylated, the enzyme itself is not subject to Edman Degradation
• Protein sequencing can follow Edman degradation without the need for an extra purification step

 

Pfu Pyroglutamate Aminopeptidase

• Removes pyroglutamic acids from the N-termini of proteins and peptides
• Enabling protein sequencing following Edman degradation

 

Documents and resources

For more information visit producer's website.