Introduction:
One of the most common mass spectrometry–based proteomic methodologies used to day is the bottom up protein identification. First, the protein has to be digested into peptides using a protease. Then, the peptide will be submitted to the URMC Proteomics Resource and Laboratory for analysis using LC-MS/MS tandem mass spectrometry. In this approach, the peptides will be separated and introduced into a mass spectrometer using reversed phase liquid chromatography (RC-LC). This spectrometer will measure the mass and charge of each peptide. Then, the peptide will be isolated and fragmented along the backbone to generate tandem of mass spectrum. The peptide MS/MS spectra will then be searched against a known protein database in order …show more content…
Protease plays in important role in processing the protein for proteomic analysis. Only a few proteases that are useful for proteomic analysis. For instance, trypsin and chymotrypsin are often used to cleave proteins at selective amino acid residues. Trypsin cleaves on the C-terminal side of the lysine and arginine amino acids, whereas chymotrypsin cleaves after the aromatic amino acids. Trypsin exhibits higher activity and narrower specificity compared to chymotrypsin. When the lysine reside is modified or flanked by proline, cleavage of trypsin is blocked (1). Since trypsin has higher activity, it was used in this lab to digest the …show more content…
A concise list of steps for preparing a sample for MS analysis is listed below.
Step 1. The E. coli pellet with GST-CamK1 construct was resuspended Phosphate Buffered Saline (PBS).
Step 2. Sonication was used to lysate the cells. Then, Triton X-100* was added the solution.
*Triton X-100 helps solubilize the proteins in cell lysate, but it has to be removed from the sample before introducing the sample into the mass spectrometer.
Step 3. The lysate GST- CamK1 sample was collected after centrifugation.
Step 4. The extract resin mix had to be washed with PBS buffer and centrifuged. The resin sample was collected.
Step 5. The AmBic buffer was first added. Then, the DTT was added to the obtained affinity resin slurry in order to reduce the disulfide bonds of the protein. The mixture was incubated.
Step 6. The IA was added to alkylate the cysteine residue. The solution was mixed and incubated in the dark*.
* Iodoacetic acid is light
One of the most common mass spectrometry–based proteomic methodologies used to day is the bottom up protein identification. First, the protein has to be digested into peptides using a protease. Then, the peptide will be submitted to the URMC Proteomics Resource and Laboratory for analysis using LC-MS/MS tandem mass spectrometry. In this approach, the peptides will be separated and introduced into a mass spectrometer using reversed phase liquid chromatography (RC-LC). This spectrometer will measure the mass and charge of each peptide. Then, the peptide will be isolated and fragmented along the backbone to generate tandem of mass spectrum. The peptide MS/MS spectra will then be searched against a known protein database in order …show more content…
Protease plays in important role in processing the protein for proteomic analysis. Only a few proteases that are useful for proteomic analysis. For instance, trypsin and chymotrypsin are often used to cleave proteins at selective amino acid residues. Trypsin cleaves on the C-terminal side of the lysine and arginine amino acids, whereas chymotrypsin cleaves after the aromatic amino acids. Trypsin exhibits higher activity and narrower specificity compared to chymotrypsin. When the lysine reside is modified or flanked by proline, cleavage of trypsin is blocked (1). Since trypsin has higher activity, it was used in this lab to digest the …show more content…
A concise list of steps for preparing a sample for MS analysis is listed below.
Step 1. The E. coli pellet with GST-CamK1 construct was resuspended Phosphate Buffered Saline (PBS).
Step 2. Sonication was used to lysate the cells. Then, Triton X-100* was added the solution.
*Triton X-100 helps solubilize the proteins in cell lysate, but it has to be removed from the sample before introducing the sample into the mass spectrometer.
Step 3. The lysate GST- CamK1 sample was collected after centrifugation.
Step 4. The extract resin mix had to be washed with PBS buffer and centrifuged. The resin sample was collected.
Step 5. The AmBic buffer was first added. Then, the DTT was added to the obtained affinity resin slurry in order to reduce the disulfide bonds of the protein. The mixture was incubated.
Step 6. The IA was added to alkylate the cysteine residue. The solution was mixed and incubated in the dark*.
* Iodoacetic acid is light