|
| Status |
Public on Jun 21, 2005 |
| Title |
Plasma membrane from Arabidopsis collected from two-phase partitioning (Sample 1) |
| Sample type |
protein |
| |
|
| Channel 1 |
| Source name |
Plasma Membrane
|
| Organism |
Arabidopsis thaliana |
| Characteristics |
PM enriched from two-phase partitioning
|
| Treatment protocol |
Sample was digested using porcine trypsin in 18O enriched water.
|
| Growth protocol |
Arabidopsis thaliana ecotype Columbia was grown at 22°C in 24 hour light in liquid culture
|
| Extracted molecule |
protein |
| Extraction protocol |
Arabidopsis thaliana ecotype Columbia was grown at 22°C in 24 hour light in liquid culture consisting of: 0.5% (w/v) MES (pH 5.7), 2.15% (w/v) Murashige and Skoog salts (Sigma), and 1% (w/v) sucrose. At two weeks of age whole seedlings were harvested.
|
| Label |
18O
|
| |
|
| Channel 2 |
| Source name |
Microsomes (PM-depleted)
|
| Organism |
Arabidopsis thaliana |
| Characteristics |
Microsomes enriched from two-phase partitioning
|
| Treatment protocol |
Sample was digested using porcine trypsin in 16O (natural abundance) enriched water.
|
| Growth protocol |
Arabidopsis thaliana ecotype Columbia was grown at 22°C in 24 hour light in liquid culture
|
| Extracted molecule |
protein |
| Extraction protocol |
Arabidopsis thaliana ecotype Columbia was grown at 22°C in 24 hour light in liquid culture consisting of: 0.5% (w/v) MES (pH 5.7), 2.15% (w/v) Murashige and Skoog salts (Sigma), and 1% (w/v) sucrose. At two weeks of age whole seedlings were harvested.
|
| Label |
16O
|
| |
|
| |
| Description |
18O Isotopic labeling experiments conducted in order to identify proteins with PM localization.
|
| Data processing |
Following digestion, peptides were separated via strong cation exchange and analyzed via RP LC MS. The raw data from each LC-MS analysis was dumped to text files using DataBridge 4.0 (Waters) and all further processing was done using Mathematica (Wolfram Research) and software programs written in C++ using Visual Studio 6.0 (Microsoft) and perl. EICs for the zero, one, and two incorporation events were extracted, 4.5 minutes wide centered on the sequencing event for all relavent peptides smoothed using a Savitzky-Golay filter and fitted to a Gaussian peak. Data values within two standard deviations of the peak center from the unsmoothed chromatograms were then used to calculate linear regressions between the monoisotopic peak and single incorporation peak as well as the monoisotopic peak and double incorporation peak in a method similar to that reported by Washburn and colleagues. Using this method, the slope of these regressions represented values for the single and double incorporation peaks that were normalized to the monoisotopic peak where the monoisotopic peak had a value of one. Peptides were only quantified if the r-squared value from both regressions was 0.8 or greater.
|
| |
|
| Submission date |
Jun 21, 2005 |
| Last update date |
Dec 12, 2005 |
| Contact name |
Michael R Sussman |
| E-mail(s) |
msussman@wisc.edu
|
| Phone |
608-262-8608
|
| Fax |
608-262-6748
|
| Organization name |
University of Wisconsin
|
| Department |
Biotechnology Center
|
| Lab |
Sussman
|
| Street address |
425 Henry Mall
|
| City |
Madison |
| State/province |
WI |
| ZIP/Postal code |
53706 |
| Country |
USA |
| |
|
| Platform ID |
GPL2543 |
| Series (1) |
| GSE2830 |
Identification of PM-localized proteins |
|
