Endoproteinase Asp-N from Pseudomonas fragi ... - Sigma-Aldrich

ENDOPROTEINASE ASP-N
Suitable for Protein Sequencing
and Peptide Mapping
Product Code P 3303
Storage Temperature 2-8 °C
TECHNICAL BULLETIN
CAS# 9001-92-7
EC: 3.4.24.33
Product Description
Endoproteinase Asp-N is a metallo endoprotease,
isolated from a mutant strain of Pseudomonas fragi,
which hydrolyzes peptide bonds on the N-terminal side
of aspartic and cysteic acid residues. 1-3
Endoproteinase Asp-N is HPLC purified, resulting in a
product that is suitable for proteomic work. In 100 mM
NH 4 HCO 3 , pH 8.5, or 100 mM Tris HCl, pH 8.5, Asp-N
specifically cleaves peptide bonds on the N-terminal
side of aspartic and cysteic acid residues. 1-3 Asp-N is
used in proteomics for peptide mapping and protein
sequence work due to its highly specific cleavage of
peptides resulting in a limited number of peptide
fragments. 1-6
Endoproteinase Asp-N has an average molecular
weight of 24.5 kDa and a pH optimum between pH 6.0
and 8.5. 1
Vial content: 2 µg of lyophilized Asp-N containing Tris-
HCl.
Precautions and Disclaimer
Self digestion may occur if temperatures above 37 °C
are used. Asp-N retains most of its activity in 2.0 M
urea, 1.0 M guanidine HCl or 0.1% SDS. 1,6 A known
peptide such as glucagon should be run as a control for
all experiments.
Preparation Instructions
Reconstitute the lyophilized product in 50 µl water. The
protease will be in a solution containing 10 mM Tris-
HCl, pH 8.0.
Storage/Stability
The lyophilized powder is stable for at least one year if
stored desiccated at 2-8 °C. After reconstitution in
water, frozen aliquots can be stored for several
weeks. 1,3
Procedure
For peptide or protein digestion, a ratio between 1/50
and 1/200 (w/w) of enzyme to substrate is
recommended. Dissolve the peptide or protein to be
digested in 100 mM NH 4 HCO 3 , pH 8.5, or 100 mM Tris
HCl, pH 8.5. Recommended incubation time is
between 2 and 18 hours at 37 °C depending on the
enzyme to substrate ratio. Endoproteinase Asp-N may
also be used for in gel digestions of proteins. 7-10
Results
The suitability of this product is demonstrated by
digestion of glucagon (Product Code G 7774) as
described in Figure 1. The sequence of glucagon is:
Absorbance, 214 nm (mAu)
HSQGTFTSDYSKYLDSRRAQDFVQWLMNT
1000
800
600
400
200
7.63
12.04 15.65 22.92
0
0 5 10 15 20 25
Time (min)
Figure 1. SUITABILITY ASSAY OF ASP-N. Glucagon (40 µg) was
digested with 2 µg of Asp-N for 18 hours at 37 °C in 100 mM
NH 4HCO 3, pH 8.5. A 20 µg aliquot was separated on a Supelco
Discovery C 18 column (25 cm x 4.6 mm, 5 micron, Product Code
504971) using a 20 minute linear gradient from 5-50% B at 0.7 ml/min
with UV detection at 214 nm and by mass spectrometry. Solvent A:
0.1% (v/v) TFA in water. Solvent B: 0.08% (v/v) TFA in acetonitrile.

The Asp-N proteolytic fragments were identified as
follows:
Retention Time (min) Mass (Da) Fragment
7.63 731.3 Asp(15)-Gln(20)
12.04 863.3 His(1)-Ser(8)
15.65 787.3 Asp(9)-Leu(14)
22.92 1152.3 Asp(21)-Thr(29)
During the 18 hour digestion only the expected peptides
were generated with no indication of other major
proteolytic activity.
References
1. Drapeau, G.R., J. Biol. Chem., 255, 839-40, (1980).
2. Wilson, K.J. et al., Methods in Protein Sequence
Analysis: Proceedings of the 7th International
Conference, Berlin, July 3-8, 1988, Wittmann-
Liebold, B. (ed.), Springer-Verlag, (New York,
1989), p 310.
3. Ponstingl, H. et al., Advanced Methods in Protein
Microsequencing Analysis, Wittmann-Liebold, B. et
al., (eds.), Springer-Verlag, (New York, 1986)
p 316.
4. Cooper, J.A. et al., J. Biol. Chem., 259, 7835-41,
(1984).
5. Guild, B.C. and Strominger, J.L., J. Biol. Chem.,
259, 9235-40, (1984).
6. Aitken, A. et al., Protein Sequencing: A Practical
Approach, Findlay, J.B.C. and Geisow, M.J. (eds.),
IRL Press, (Oxford, 1989), p 43.
7. Eckerskorn, C. and Grimm, R., Electrophoresis, 17,
899-906, (1996).
8. Scheler, C. et al., Electrophoresis, 19, 918-27,
(1998).
9. Jimenez-Asensio, J., et al., J. Biol. Chem., 274,
32287-94, (1999).
10. Jeno, P., et al., Anal. Biochem., 224, 75-82, (1995).
SIGMA HPLC Purified Products:
Product Package Product
Name Size Code
Carboxypeptidase Y 25 µg C 4046
α-Chymotrypsin 25, 100 µg C 6423
Endoproteinase Arg-C 5 µg P 6056
Endoproteinase Asp-N 2 µg P 3303
Endoproteinase Glu-C 25 µg P 6181
Endoproteinase Lys-C 5 µg P 3428
Leucine aminopeptidase 50 µg L 9776
Trypsin 100 µg T 8658
Insulin Chain B, Oxidized 100 µg I 1764
Melittin 100 µg M 1407
Glucagon 100 µg G 7774
α-Melanocyte 1, 5 mg M 4135
Stimulating Hormone
Titles of Related Books (Product Code):
MASS SPECTROMETRY OF PROTEINS AND
PEPTIDES: Methods in Molecular Biology (M 7059)
MICROCHARACTERIZATION OF PROTEINS
(M 9807)
NEW METHODS IN PEPTIDE MAPPING FOR THE
CHARACTERIZATION OF PROTEINS (Z36,931-4)
A PRACTICAL GUIDE TO PROTEIN AND PEPTIDE
PURIFICATION FOR MICROSEQUENCING
(Z35,034-6)
SEQUENCING OF PROTEINS AND PEPTIDES:
Laboratory Techniques in Biochemistry and
Molecular Biology, 2nd Ed. (S 2521)
PROTEOLYTIC ENZYMES: A Practical Approach
(P 4926)
PROTEIN STRUCTURE: A Practical Approach
(Z37,548-9)
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
OF PEPTIDES AND PROTEINS: Separation,
Analysis and Conformation (H 8147)
HPLC OF MACROMOLECULES: A Practical Approach
(H 3409)
PROTEIN SEQUENCING PROTOCOLS: Methods in
Molecular Biology (Z37,395-8)
LKB 6/01
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