The primary structure of the protein must be known!
Samples containing a given protein either obtained after expression in different expression systems (e.g. prokaryotic vs. eukaryotic), or before and after induction, or before and after in vitro modification (e.g. phosphorylation) might be compared.

You provide us with a) or b):

a) Homogenous protein per sample, obtained by excision of bands 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 bands which were excised from the gel, and specify the molecular weight marker and the estimated molecular weight of the proteins of interest.

b) At least 25 µg of each purified native protein sample(ammonium sulfate precipitate or lyophilized protein). Please, enclose a photo of an analytical SDS-PAGE, mention the %age of the gel, clearly mark the bands which correspond to the purified proteins, and specify the molecular weight marker and the estimated molecular weight of the proteins of interest. Recommend a suitable buffer for dialyzing (ammonium sulfate precipitate) or dissolving (lyophilized material) the proteins. 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 samples (organism, tissue, cell fraction etc.), and assumption which protein modification is anticipated.

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

 

Our service:

1. (In gel) trypsination of the protein (bands) of interest (Protein is cleaved at arg and lys sites into peptides)
2. MALDI-TOF Mass Spectometry analysis of the tryptic peptides
3. Peptides carrying posttranslational modifications are identified by comparing their actual mass with their theoretical mass peak by peak. This process is carried out by trained 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.
4. A comparison list of modifications for the different protein samples is delivered.

Order Information:

Cat. Nr. CPPM01: Comparison of Posttranslational Modifications of a Protein by MALDI-TOF-MS, proteins shipped in protein gel bands
Price: 495 Euro per sample (minimum: 2 samples)

Cat. Nr. CPPM02: Comparison of Posttranslational Modifications of a Purified Protein by MALDI-TOF-MS
Price: 720 Euro per sample (minimum: 2 samples)

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.

Acetylation is the binding of an acetyl group to an amino acid. This adds about 42 Da to the acetylated peptide. If the acetylation affects the N-terminus of the peptide, the first amino acid methionine can be eliminated. This causes a loss of 131.1 Da corresponding to the mass of the methionine, but the concomitant addition of the acetyl group reduces this mass loss to about 89 Da. In the example shown above the protein purified from E. coli is not acetylated (as expected) and contains the first methionine. Accordingly, the mass corresponding to the unmodified peptide fragment T1-16 (m/z 1913.8) was detected. The peak corresponding to the T1-16 fragment was not measured in the digest of the same protein expressed in either yeast or human skeletal muscle. Instead, the peak corresponding to the loss of methionine and the addition of an acetyl group (on the threonine 2) was detected (m/z 1825.2).