Difference between revisions of "Lidstrom:13C Incorporation Into Protein - Data Analysis"

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(Our protocol)
(Our protocol)
Line 8: Line 8:
### serine is a good 1st choice.
### serine is a good 1st choice.
### after serine, look at ____.
### after serine, look at ____.
# Chromatogram --> extract ion chromatogram
## Enter time range that includes your expected retention time and a few minutes of buffer.
==Data Analysis from Martin==
==Data Analysis from Martin==

Revision as of 15:54, 20 December 2012

Back to Protocols

Our protocol

  1. Open Start --> Programs --> MSD ChemStation --> Instrument #1 --> Instrument #1 Data Analysis
  2. Load the compound library from Z:\Common\Software\ChemStation Library/NIST08.L
  3. Chose an ion to look for
    1. Chose an amino acid, the use Yanfen's table to see the unique ion retention time and m/z.
      1. serine is a good 1st choice.
      2. after serine, look at ____.
  4. Chromatogram --> extract ion chromatogram
    1. Enter time range that includes your expected retention time and a few minutes of buffer.

Data Analysis from Martin

  • Open Enhanced Data Analysis (ChemStation) from the desktop
  • Mouse Actions
    • Left button zoom, Double click with left resets view
    • Right button action

Open File

  • Open a file
    • Or if your file is running, take a snapshot of the file and you can analyze that while the run is still going

Looking at your data

  • To get an ion chromatogram for a peak: Draw Rt. button over part of the peak to get the ion chromatogram. Only the width of the box matters in terms of which ions will appear in the ion chromatogram
  • To identify compounds in the ion chromatogram: Right button double click on the ion chromatogram to have Chemstation try to identify the compounds. This will turn up a list of possible matches. Need to go through each peak an look @ a list of possible candidates.

• ex) proline matches peak, but weird 283 peak ○ extract ion chromatogram for the different ions in the 283 and 253 peak --> can see to overlapping peaks • Best ion to look for is the M-57 --> shift by number of labelled carbons • select two areas, then go to spectrum --> subtract to do background substitution

  • Go to spectrum tabulate to get a list of intensities. Copy ions to spreadsheet.

Data Analysis from Yanfen

Open Chemstation From menu--> chromatograms--> Extract ion chromatograms Use data from standard run (Yanfen said she'd send us Bo's data. This is a confirmation of the data from the table in the paper listing unique ions for each compound, std data does specify retention times) Put in time range and unique ions into form --> get mid point of peak at retention time to get average intensity (rt click drag box) --> of peaks in the ion chromatogram use the higher intensity peak usually the M-57. ex) for Ser use 390 not 432

Internal standard +4 all labeled C + 1 N labeled. --> normalized peak intensity to this

Look for N+3 --> 393 instead of 390.

Example Ser ion chromatogram 447 --> M-15 (loss of Ch4) 432 --> 390 M-57 highest abundance

Select peak --> go ion spectrum --> right double click

11/19 2:30pm --> 12am 11/20 Agilent 5975 GCMS