Asp-N Protease: Applications Update

TrypsinLysC Page 2

Asp-N, Sequencing Grade, is an endoproteinase that hydrolyzes peptide bonds on the N-terminal side of aspartic and cysteic acid residues: Asp and Cys. Asp-N activity is optimal in the pH range of 4.0–9.0. This sequencing grade enzyme can be used alone or in combination with trypsin or other proteases to produce protein digests for peptide mapping applications or protein identification by peptide mass fingerprinting or MS/MS spectral matching. It is suitable for  in-solution or in-gel digestion reactions.

The following references illustrate the use of Asp-N in recent publications:

Protein sequence coverage

  1. Jakobsson, M et al. (2013)  Identification and characterization of a novel Human Methyltransferase modulating Hsp70 protein function through lysine methylation. J. Biol. Chem. 288, 27752–63.
  2. Carroll, J. et. al. (2013) Post-translational modifications near the quinone binding site of mammalian complex I.  J. Biol. Chem. 288, 24799–08.

Glycoprotein analysis

  1. Siguier, B. et al. (2014) First structural insights into α-L-Arabinofuranosidases from the two GH62 Glycoside hydrolase subfamilies. J. Biol. Chem. 289, 5261–73.
  2. Vakhrushev, S. et al. (2013) Enhanced mass spectrometric mapping of the human GalNAc-type O-glycoproteome with SimpleCells. Mol. Cell. Prot. 12, 932–44.
  3. Berk, J. et al. (2013) . O-Linked β-N- Acetylglucosamine (O-GlcNAc) Regulates emerin binding to autointegration Factor (BAF) in a chromatin and Lamin B-enriched “Niche”.  J. Biol. Chem. 288, 30192–09.

Phosphoprotein analysis

  1. Roux, P. and Thibault, P. (2013) The Coming of Age of phosphoproteomics –from Large Data sets to Inference of protein Functions. Mol. Cell. Prot. 12, 3453–64.

Increased protein identification using Pseudomonas and Lysobacter Lys-C proteases

Alternate Proteases CoverOwing to efficient proteolysis and particular advantages of trypsin-generated peptides for mass spectrometry analysis, trypsin is the most widely used proteomic protease. Recently, however, Lys-C has been increasingly used as either a trypsin alternate or as supplement. Its increasing favor is largely due to its ability to perform proteolytic digestion under protein denaturing conditions, an attribute that can greatly extend the observable proteome.

Lys-C is found in number of bacterial hosts with Lysobacter enzymogenes being used as a most popular source of commercially available Lys-C. We have now developed a recombinant form of Lys-C from Pseudomonas aeruginosa. We have compared performance of Pseudomonas and Lysobacter Lys-C.
Surprisingly, we found difference between Pseudomonas and Lysobacter Lys-C proteases on peptide level. The peptides generated by the proteases had much smaller overlap (25%) than typically observed between runs for the same sample indicating different bias toward lysine cleavage sites for Pseudomonas and Lysobacter Lys-C.
The proteases might have different proteolytic mechanisms. In fact, difference in proteolytic mechanisms is not unexpected considering the limited homology between these two proteases
Therefore we recommend combined digestion with Pseudomonas and Lysobacter Lys-C to maximize peptide and protein identification.

For a detailed technical review of these two protease visit: