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| Status |
Public on Sep 20, 2005 |
| Title |
MMP qualitative and quantitative mass spectrometry |
| Platform organisms |
Methanococcus maripaludis; Methanococcus maripaludis S2 |
| Sample organism |
Methanococcus maripaludis |
| Experiment type |
Other
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| Summary |
Methanococcus maripaludis is a methanogenic archaeon. Within its genome, there are two operons for membrane associated hydrogenases, eha and ehb. To investigate the regulation of ehb on the cell, an S40 mutant was constructed in such a way that a portion of the ehb operon was replaced by pac cassette in the wild type parental strain S2 (done by Whitman's group at the University of Georgia). The S40 and S2 strains were grown in 14N and 15N media with acetate separately. A biological replicate was made by switching the media. Mass spectrometry based quantitative proteomics were done on the mixtures to investigate the differences in expression patterns between S40 and S2.
Keywords: isotope labeling mass spectrometry, quantitative proteomics
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| Overall design |
Methanococcus maripaludis is a methanogenic archaeon. Within its genome, there are two operons for membrane associated hydrogenases, eha and ehb. To investigate the regulation of ehb on the cell, an S40 mutant was constructed in such a way that a portion of the ehb operon was replaced by pac cassette in the wild type parental strain S2 (done by Whitman's group at the University of Georgia). Thus the S40 mutant can be selected from the S2 background by puromycin screening. The S40 and S2 strains were grown in 14N and 15N media with acetate separately. A biological replicate was made by switching the media, i.e. "flipping" the isotopes. Samples were mixed together then, after that, the mixed sample was separated to soluble part (CE part) and particulate part (PT part).
Trypsin digestion was done after treating the sample with RapiGest, DTT and iodoacetamide. Then, an YMC C18 column was used to make fractions from F1 to F5. A two dimensional capillary HPLC column was used to load the sample. The capillary column contains two parts, a strong cation exchange column (SCX) and a reverse phase part (RP), which is an Aqua C18 column. Seven different salt concentrations of ammonium acetate were used to elute the sample from SCX to the C18. Then, a gradient of acetonitrile was performed to elute the peptides from the C18 part. Electrospray ionization was done at 2.4 kv. MS1 and MS2 spectra were collected on a ThermoFinnigan LCQ. Raw files were processed by SEQUEST software and DTASelect v1.9. The Xcorr criteria used in DTASelect were 1.9 for singly charged, 2.0 for doubly charged, 3.3 for triply charged. The data were processed by SEQUEST separately using 14N and 15N amino acid modification parameters. So after DTASelect, two sets of protein identification tables were created from each dataset.
A program calculating the protein expression ratios in S40 and S2 was written by Fred Taub in the FileMaker 6.0 environment. The scheme of the program is shown in Fig.2. First, from the DTASelect-filter.txt file, for each CID scan file, calculate the corresponding isotope ion's m/z. Then, go back to the raw file data, finding the full scan from which the CID scan came. Compare the intensity of the full scans right before and behind this full scan in the reconstructed ion current chromatogram (RIC chromatogram). Use the most intense one among the three for the calculation of protein's ratio. Then, for each peptide from the same or very close full scan, compare the calculated ratio both from 14N DTASelect-filter.txt and 15N DTASelect-filter.txt files. Assign confidence values A, B, or C to each ratio pair by the following rules: if the ratios from both DTASelect-filter.txt files are the same, assign them to confidence A level; if they are different, assign B; if one side is missing, then assign C value. Then, each protein's 14N/15N ratio was calculated as the mean value of all unique peptides identified for the protein that have the highest confidence value.
After the protein ratios were produced for each sample, CE1, CE2, PT1 and PT2, all the data were normalized and combined to make a final report of the expression pattern of S40/S2. All the ratios before assigning confidence values were used in this calculation. The detection of outliers was done in two steps. For the first stage, Dixon's Q-test was used; then, at the second stage, a MAD modified z-score test (18) with >3.5 cutoff was used. By Dixon's Q-test, 300 records were detected as outliers from 21524 records, which is 0.72%. Then, by MAD modified z-score test, 1520 records were detected as outliers from the remaining 21224 records, which is 7.2%.
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| Contributor(s) |
Hackett M, Zhang Y, Wang T, Xia Q |
| Citation(s) |
16452419, 16489187, 17124542 |
| Submission date |
Jun 03, 2005 |
| Last update date |
Mar 25, 2020 |
| Contact name |
Murray Hackett |
| E-mail(s) |
MHACKETT@U.WASHINGTON.EDU
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| Phone |
206-616-8071
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| Organization name |
University of Washington
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| Department |
Chemical Engineering
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| Lab |
Hackett
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| Street address |
HSB H330 1959 NE Pacific Street
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| City |
Seattle |
| State/province |
WA |
| ZIP/Postal code |
98195 |
| Country |
USA |
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| Platforms (2) |
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| Samples (2) |
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| Relations |
| BioProject |
PRJNA92213 |
| Supplementary data files not provided |
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