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Identification of posttranslational protein modifications
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Download MALDI-TOF
Service Information
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The primary
structure of the protein must be known!
You provide
us with: a) or b):
a)
Homogenous protein in an excised band from a dried SDS-gel (stained
with Coomassie Blue or Silver, preferably without fixation). Please
enclose a gel photo. Please mention the %age of the gel, clearly
mark the band which was excised from the gel, and specify the
molecular weight marker and the estimated molecular weight of the
protein of interest.
b) At
least 25 µg purified native protein (ammonium sulfate precipitate or
lyophilized protein). Please, enclose a photo of an analytical
SDS-PAGE, mention the %age of the gel, clearly mark the band which
corresponds to the purified protein, and specify the molecular
weight marker and the estimated molecular weight of the protein of
interest. Recommend a suitable buffer for dialyzing (ammonium
sulfate precipitate) or dissolving (lyophilized material) the
protein. If this is not a standard buffer, add your buffer in
sufficient amount (esp. for dialysis). Mention special requirements
of your protein with respect to solubility and stability (especially
important for membrane proteins).
Required
Information:
Protein identity
(incl. database entry number) and source of the protein (organism,
tissue, cell type, sub-cellular fraction etc.).
Please ship the
protein without cooling (dried gel bands, lyophilized protein) or on
blue ice (ammonium sulfate precipitate) to:
Gentaur
Avenue de l'Armée 68/B4
B-1040 Brussels BELGIUM
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Our service:
- (In gel)
trypsination of the protein (band) of interest (protein is cleaved
at arg and lys sites into peptides)
- MALDI-TOF Mass
Spectometry analysis of the tryptic peptides
- Peptides
carrying posttranslational modifications are identified by comparing
their actual mass with their theoretical mass peak by peak. This
process is carried out by experienced scientists. A list of
experimental peptides that correspond to unmodified peptides is
generated, then a list of experimental peptides that match to
peptides which carry modifications of cystein residues, methionine
residues, or other modification as documented in SWISS-PROT is
created. In order to find novel modifications, the mass differences
between all experimental peptides and all theoretical peptides are
calculated. If a mass difference corresponds to the mass of any
known modification the peptide is classified as potentially
modified. Within the peptide the potential amino acid(s) that may
carry the modification in question is/are suggested.
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Order
Information:
Cat. Nr.
IPM01: Identification of Posttranslational Protein Modification
by MALDI-TOF-MS, protein shipped in protein gel band
Price: 650 Euro
Cat. Nr.
IPM02: Identification of Posttranslational Protein Modification
by MALDI-TOF-MS, purified protein provided
Price: 720 Euro
Please note:
In case the protein amount or purity was not sufficient to perform
MALDI-TOF MS, a set up fee of € 175 is charged.
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Example: |
Phosphorylation
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Phosphorylation of
proteins is catalysed by protein kinases. In most cases this
modification has a regulatory role, also a stabilisation effect is
possible. The phosphorylation of proteins adds about 80 Da to the
protein mass resulting from the mass of the added phosphate group. This
mass difference can be detected by the MALDI-TOF MS when mass spectra
from the phosphorylated and dephosphorylated (or unphosphorylated)
sample are compared. The peptide fragment carrying the phosphorylation
is 80 Da larger and is absent in the dephosphorylated sample. The
example below illustrates the mass spectra from a phosphorylated and
dephosphorylated sample. The phosphorylation of the peptide with m/z
1414.2 Da leads to a phosphorylated fragment (m/z 1494.4 Da). The peak
corresponding to the phosphorylated fragment was not detected after
phosphatase treatment.
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Last modified:
05/19/16
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