For research use only. Not for use in diagnostic procedures.
Using the TripleTOF™ 5600 and 6600 Intact Protein Analysis Script
The Intact Protein Analysis script is used whenever proteins with MW > ~ 6kDa are analyzed on the 5600. The script makes modifications that improve the analysis of both small and large proteins and should be used in all cases.
THIS IS A PRE-BETA VERSION OF THE SCRIPT AND IS TO BE USED ONLY BY ABSCIEX PERSONNEL ONLY. IT IS NOT FOR CUSTOMER DISTRIBUTION.
Installation of the Script:
Using the Script:Access the script in the Scripts menu in Analyst under ‘IntactProteinMode’. The interface will appear and will indicate that Intact Protein Mode is ‘Off’. In order to activate Intact Protein Mode click the ‘IPM Activate’ button. The user also has the option to have the CEM voltage decreased automatically by 100V in TOFMS mode only by selecting the checkbox (must be checked prior to clicking IPM Activate). This decreases the signal from singly charged background while having little effect on the signal for multiply charged species.
- Close Analyst
- Copy the files IntactProteinMode.dll and Intact Protein Analysis Settings.txt to the ‘Processing Scripts’ directory in the API Instrument project.
- Re-open Analyst
Once the IPM Activate button is pressed the following warning will appear instructing the user to restart AnalystService in order for the changes to take effect:
Once activated the UI will update the Intact Protein Mode status to ‘On’. The script can now be closed.Deactivation:Intact Protein Mode is deactivated by re-opening the script and clicking on ‘IPM Disable’. This will be followed by a message to restart Analyst Service in order for the original settings to be restored.
How will I know it is working?
When analyzed using default settings on the 5600 proteins will show certain easily recognized artifacts. Smaller proteins for which isotopic resolution is possible will show a marked suppression of the low m/z peaks in the isotope cluster. This is shown below for horse myoglobin (5pmol injection) for a single scan spectrum near the apex of the LC peak:
With Intact Protein Mode activated the isotope cluster will not show this artifact and will have a Gaussian shape.
For larger proteins (MW> ~25 kDa) the use of default settings may result in splitting on the low m/z side of the peak as well as consistent dropping out of the signal in the baseline. This is shown below for bovine apo-transferrin (10 pmol injection) for a single scan spectrum near the maximum of the LC peak:
Activation of Intact Protein Mode will generally result in considerably improved signal-top-noise for the protein peaks, as well as elimination of the peak splitting and signal dropouts:
Time Bins to Sum:
For proteins for which isotopic resolution is desired (MW<20 kDa) it is recommended that a Time Bins to Sum value of 1 be selected. For larger proteins a value of 10 is recommended.