Identification of a protein by peptide mass fingerprinting. A protein with an ap
ID: 177980 • Letter: I
Question
Identification of a protein by peptide mass fingerprinting.
A protein with an apparent molecular weight of 59.6 kDa was isolated from a 2D-gel and submitted to in-gel trypsin digestion. From the MALDI-TOF mass spectrum, the following list of 8 masses was established:
MH+ (Da, monoisotopic)
560.31
575.31
602.36
733.42
989.52
1428.59
1442.59
1826.91
1928.97
2217.06
2437.17
Access the Mascot search site http://www.matrixscience.com/cgi/search_form.pl?FORMVER=2&SEARCH=PMF.
Copy and paste the eight masses. Before you start the search enter your e. mail address and select the following parameters:
- Database: SwissProt
- Taxonomy: Bacteria (Eubacteria)
- Allow up to: 1 missed cleavage
- Peptide tolerance: 1 Da
Based on the information included above define the following parameters: enzyme, protein mass, and mass values. Do not change the other parameters.
a. Identify the protein with the best score.
b. Briefly explain why you have likely identify the correct protein.
c. In the concise summary report, select the protein with the highest score (click on the blue access number) to get the sequences of the identified peptides. What is the sequence of the 1428.59 Da peptide?
d. One experimental mass does not match any of the theoretical masses. Independent experiments suggest that the unmatched mass corresponds to a peptide that contains one of the following post-translational modifications.
- Biotinylation of a peptide
- Methylation of a glutamic acid (E)
-Methylation of the C-terminal end of a peptide
To determine which type of modification affected one of the peptide, repeat the search after you have selected the Biotin (K), Methyl (C-term) or Methyl (DE) “variable modification” (scroll down the list of possible modifications, select ONE of the modification and then click on the arrowhead (<) to show the selected modification in the “variable modification” window). Briefly explain your result. Selecting the 3 modifications at the same time will not give you any results. You need to repeat the search 3 times, one per modification. Note that on my computer, I cannot scroll down the lists of possible modifications with my mouse, I need to use the down (or up) arrow on my keyboard.
Which one of the peptides was modified and what was the post-translational modification?
Explanation / Answer
Answers:
a. MCP2_SALTY Mass: 59577 Score: 108 Expect: 5.3e-06 Matches: 10 Methyl-accepting chemotaxis protein II OS=Salmonella typhimurium (strain LT2 / SGSC1412 / ATCC 700720) GN=tar PE=1 SV=3.
Protein View: MCP2_SALTY
Methyl-accepting chemotaxis protein II OS=Salmonella typhimurium (strain LT2 / SGSC1412 / ATCC 700720) GN=tar PE=1 SV=3
c. Highest score : MCP2_SALTY Mass: 59577 Score: 108 Expect: 5.3e-06 Matches: 10 Methyl-accepting chemotaxis protein II OS=Salmonella typhimurium (strain LT2 / SGSC1412 / ATCC 700720) GN=tar PE=1 SV=3
Sequence: R.MMMDASNQQSSAK.T
d. Biotinylation of a peptide
RNH_RICTY Mass: 20767 Score: 44 Expect: 13 Matches: 6 Ribonuclease H OS=Rickettsia typhi (strain ATCC VR-144 / Wilmington) GN=rnhA PE=3 SV=1
Methylation of a glutamic acid (E)
SYA_BACC1 Mass: 113304 Score: 53 Expect: 1.7 Matches: 9 Alanine--tRNA ligase OS=Bacillus cereus (strain ATCC 10987 / NRS 248) GN=alaS PE=3 SV=1
Methylation of the C-terminal end of a peptide
YIDC_ALKEH Mass: 68556 Score: 50 Expect: 3 Matches: 9 Membrane protein insertase YidC OS=Alkalilimnicola ehrlichii (strain ATCC BAA-1101 / DSM 17681 / MLHE-1) GN=yidC PE=3 SV=1
Database: SwissProt Score: 108 Expect: 5.3e-06 Monoisotopic mass (Mr): 59577 Calculated pI: 5.07 Taxonomy: Salmonella enterica subsp. enterica serovar Typhimurium str. LT2Related Questions
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