ASTM E1759-95
(Test Method)Standard Test Method for Isoaspartic Acid in Proteins Method for the Determination of Asparagine Deamidation Products
Standard Test Method for Isoaspartic Acid in Proteins Method for the Determination of Asparagine Deamidation Products
SCOPE
1.1 This test method covers the determination of isoaspartic acid residues in a protein or peptide sample. This test method is applicable for the determination of isoaspartic acid residues in a sample in the range of 2.5-50 [mu]mol/L. Higher concentrations can be determined following dilution. The reported lower range is based on single-operator precision.
1.2 The values stated in SI units are to be regarded as the standard.
1.3 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.
General Information
Relations
Standards Content (Sample)
NOTICE: This standard has either been superceded and replaced by a new version or discontinued.
Contact ASTM International (www.astm.org) for the latest information.
Designation: E 1759 – 95
Standard Test Method for
Isoaspartic Acid in Proteins: Method for the Determination
of Asparagine Deamidation Products
This standard is issued under the fixed designation E 1759; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (e) indicates an editorial change since the last revision or reapproval.
INTRODUCTION
The storage of proteins in aqueous solutions often results in the formation of isoaspartic acid
linkages within the polypeptide chain as a result of the deamidation of aspargine residues and the
rearrangement of aspartic acid linkages. This test measures the amount of isoaspartic acid residues in
a protein or peptide solution by the use of the enzyme protein isoaspartyl methyl transferase and
radioactive S-adenosyl-L-methionine.
1. Scope form of a co-factor that is consumed in the enzymatic reaction
of the enzyme. During the test a radiolabelled intermediate is
1.1 This test method covers the determination of isoaspartic
formed through the transfer of the labeled methyl group from
acid residues in a protein or peptide sample. This test method
S-adenosyl-L-methionine to the alpha carboxy group of isoas-
is applicable for the determination of isoaspartic acid residues
partic acid. This methylated intermediate is then degraded to
in a sample in the range of 2.5–50 μmol/L. Higher concentra-
liberate the methyl group as methanol. The methanol is then
tions can be determined following dilution. The reported lower
captured in a methanol diffusion procedure and counted.
range is based on single-operator precision.
3.2 A sample of protein is incubated with the enzyme
1.2 The values stated in SI units are to be regarded as the
protein isoaspartyl methyl transferase and radiolabelled
standard.
S-Adenosyl Methionine in a buffer that results in the accumu-
1.3 This standard does not purport to address all of the
lation of the methyl esters of isoaspartic acid residues through
safety concerns, if any, associated with its use. It is the
the enzymatic transfer of the methyl group from S-adenosyl-
responsibility of the user of this standard to establish appro-
L-methionine to isoaspartic acid sites in the protein. The
priate safety and health practices and determine the applica-
protein solution is then treated with a basic solution containing
bility of regulatory limitations prior to use.
sodium dodecyl sulfate in order to inactivate the enzyme and
2. Terminology convert the methylated isoaspartic acid residues to a succin-
imide and free methanol. The methanol is then separated from
2.1 Definitions of Terms Specific to This Standard:
the protein solution through the diffusion of the methanol to a
2.1.1 isoaspartic acid residue—indicates an aspartic acid
scintillation fluid solution. The methanol transferred to the
residue in which linkage of the polypeptide chain takes place
scintillation fluid is then determined by counting of the
through the gamma carboxyl group of the aspartic acid versus
radioactivity in the scintillation fluid.
the alpha carboxyl group that is used in the normal peptide
linkage.
4. Significance and Use
3. Summary of Test Method
4.1 Isoaspartic acid residues are generated during incuba-
tion of proteins under a wide variety of conditions in aqueous
3.1 The basis of the procedure given in this test method is
solution. Such residues are generated most commonly through
the production of radioactive methanol equal to the amount of
the deamidation of aspargine residues although some reports of
isoaspartic acid residues present in a protein sample through
isoaspartic acid formation through the rearrangement of aspar-
the action of the enzyme protein isoaspartyl methyl transferase
tic acid residues have been published.
and radiolabelled S-adenosyl-L-methionine, a radiolabelled
4.2 The presence of such residues can indicate that the
protein containing such residues has suffered damage that may
This test method is under the jurisdiction of ASTM Committee E-48 on
affect the biological activity of the protein. The precise
Biotechnology and is the direct responsibility of Subcommittee E48.02 on Charac-
terization and Identification of Biological Systems.
Current edition approved Oct. 10, 1995. Published December 1995.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.
NOTICE: This standard has either been superceded and replaced by a new version or discontinued.
Contact ASTM International (www.astm.org) for the latest information.
E1759–95
correlation between the level of isoaspartic acid content and the 8. Calibration
biological activity of the protein needs to be determined on a
8.1 Prepare 50 pmol/5 mL reference standard solution and a
case by case basis.
reaction blank solution (0 pmol/5 mL). Dilute the IsoAsp-DSIP
4.3 The test measures the level of isoaspartic acid content in
reagent provided with the ISOQUANT kit with water to create
a protein sample. This level will often be correlated with the
the reference standard solution and use the water for the
degree to which the protein has suffered deamidation at
reaction blank solution.
asparagine residues. In addition, isoaspartic acid residues can
arise on occasion through the rearrangement of aspartic acid
9. Procedure
residues. For these reasons, the level of isoaspartic acid
9.1 Determine the number of reactions that will be run in the
residues in proteins can be used as a general indication that the
test. Each test should contain a 0 and 50 pmol IsoAsp-DSIP
protein sample has suffered some level of damage and should
standard along with any unknowns. All samples and standards
not be interpreted to indicate the precise level of damage to any
are to be used in duplicate specimens.
one region within a protein without further testing.
9.2 Prepare a IsoAsp-DSIP reference standard by diluting
the IsoAsp-DSIP standard to 10 μmol in a 1.5 mL microcen-
5. Interfering Substances
trifuge tube with water and mixing by vortex for 15 s. Prepare
5.1 Sodium dodecyl sulfate and guanidine hydrochloride
at least 20 μL of diluted reference standard. Refer to the
will interfere with this test by inactivating the enzyme.
certificate of analysis provided with the IsoAsp-DSIP material
5.2 Highly acidic, basic or buffered solutions that alter the
for the exact concentration of the standard with the kit to be
pH of the reaction mixture from pH 6.2 can interfere with the
used.
assay by altering the kinetics of the enzymatic reaction used in
9.3 Calculate the amount of H-SAM stock solution needed
the test in either a positive or negative way.
in the assay. For each reaction to be run, add 1.1 μL of
S-adenosyl-L-methionine and 1.1 μCi of H-SAM to a 1.5 mL
6. Apparatus
microcentrifuge tube and add water to a final volume of 11 mL.
9.4 Prepare reaction master mix. For each reaction to be run,
6.1 Scintillation Counter.
add 11 mL of water; 11 mL of 5X reaction buffer; 11 mL of
6.2 Scintillation Vials—Scintillation vials capable of hold-
protein isoaspartyl methyltransferase; and 11 mL of H-SAM
ing at least 4.5 mL of scintillation fluid and capable of being
stock solution in a 1.5 mL microcentrifuge tube. Add the
heated to 40°C for an extended period of time without damage
materials in the order given and mix by vortex 15 s.
in the presence of scintillation fluid are used.
9.5 Place two labeled 1.5 mL microcentrifuge tubes on ice
6.3 Microcentrifuge.
for the reaction blank, the 50 pmol IsoAsp-DSIP calibration
6.4 Positive Displacement Pipettes.
standard and for each sample to be run.
9.6 Insert one sponge insert into a scintillation vial cap for
7. Reagents and Materials
every reaction that will be performed. Attach the sponge insert
7.1 Protein Isoaspartyl Methyltransferase.
to the inside of the vial cap by removing the backing on the
7.2 IsoAsp-DSIP (Delta Sleep Inducing Peptide).
sponge and attaching the adhesive site of the sponge to the
7.3 5X Reaction Buffer.
inside of the cap.
7.4 S-Adenosyl-L-Methionine.
9.7 Fill a scintillation vial to half its capacity with scintil-
7.5 Stop Solution.
lation fluid for each assay to be performed.
7.6 Sponge Inserts.
9.8 Add 10.0 mL of each unknown and reaction blank
3 3
7.7 Tritiated S-Adenosyl-L-Methionine, [ H-SAM].
sample to the appropriate labeled sample tube and place the
7.8 Scintillation Cocktail—A standard scintillation fluid
tube on ice. Add 5.0 mL of the IsoAsp-DSIP reference standard
with a flash point greater than or equal to 150°C and capable of
and 5.0 mL of water to each reference standard sample tube
use for the counting of tritiated compounds is required.
and place the tube on ice.
2 4
Promega Corp. has an exclusive license t
...
Questions, Comments and Discussion
Ask us and Technical Secretary will try to provide an answer. You can facilitate discussion about the standard in here.