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Your assignment is to prepare and submit a paper on proteomic profiling and data.
Your assignment is to prepare and submit a paper on proteomic profiling and data. In peptide mass fingerprinting (PMF), experimental data on the peptide molecular mass is compared against compared with the theoretical data that are deposited in protein data banks like the NCBInr and SwissProt databases. This is possible with the use of different software like MASCOT PMF, ProFound, and Peptide.
In this exercise, a sample was obtained from cells of cultured Rat Basophilic Leukemia (RBL 293) cells, which were grown in a medium supplemented with fetal calf serum. The cells were lysed and prepared for 2D-PAGE. The first dimension involved the separation of the proteins by IEF on a linear pH 4-7 IPG strip (horizontal), followed by SDS-PAGE on a 10% acrylamide gel (vertical). The gel was silver-stained and imaged using a densitometer. The approximate mass, M, and pI of the proteins can be seen in the image. The spots of interest, A-F, were excised from the gel, destained, reduced, and alkylated with iodoacetamide (CAM-derivative). Iodoacetamide is added to covalently modify cysteine residues to form carbamidomethylcysteine to present the formation of disulfide bridges and to ensure that the proteins are completely unfolded. After this step, proteins are cleaved by trypsin, a proteolytic enzyme that cleaves lysine and arginine except when followed by proline toward the C-terminal direction, to form peptide fragments.
The digests were analyzed by MALDI-ToF mass spectrometry by an instrument with a mass error of 50-200 ppm. The spectra obtained were processed to generate the peptide mass fingerprints and the monoisotopic peak list. This list was submitted to www.matrixscience.com which has the online Mascot PMF software that searches the SwissProt or NCBInr databases for proteins with peptides of similar masses. The search parameters used were:
Variable modifications: Oxidation (M). but depending on the results obtained, this was changed to look for higher probability scores. An increasing number of modifications will lessen the probability because of the increased number of possible peptide masses. .
