How to extracted the MS1 intensity and charge state information from DDAPASEF and diaPASEF files by timspy

[Mnago]

Hi all,
Background:
I successfully extracted the m/z and ion mobility of MS1 from DDAPASEF data by timspy. Which was followed by the link below. https://github.com/MatteoLacki/timspy
The m/z and ion mobility information could also be extracted by the MGF files which were generated after data acquisition.

Issues:

1. We used the two pipelines to extracted the same files (DDAPSEF files). The results show that 251158 ions were obtained from MGF files, while 7228723 ions from timspy. I am wondering why is there such big differences?
2. How to extract the charge state and intensity information of MS1 from DDAPASEF files and diaPASEF files by timspy?

Looking forward to your reply.
Best regards,

Winnie

[MatteoLacki]

Hi,

Ad 1. I don't know what tool is used to create these MGFs and what it does. I am sure that the number of peaks reported by TimsPy corresponds to all raw data points (.tdf_raw stores centroided peaks and we don't cut any away). Maybe your software aggregates out the mobility dimension?

Ad 2. Tables Precursors and PasefFrameMsMsInfo store the necessary information. You can get them to pandas using table2df method of any class that connects to the data.

Best wishes,

[Mnago]

Hi,

1. The MGF files were automated generated by the data acquisition process with a processing method suggested by the instrument application engineer. I will check the processing method.

2. I have extracted the precursors, ion mobility, and m/z information from .tdf files by software named DB Browser for SQLite with PasefFrameMsMsInfo and Precursors Table. But there are only part of points be extracted. The results show that 255387 ions were obtained from .tdf files by the DB browser, while 7228723 ions from timspy. I am wondering the precursor ions were extracted from DB Browser, while all ions (precursor ion and ions which are not selected for fragmentation) were extracted from TimsPy, right? if yes, How to extracted the precursor and charge state information from DDAPSEF and diaPASEF files?

Best regards,

[MatteoLacki]

Ad 1. Do you mean something like Compass HyStar?

Ad 2. TimsPy parses analysis.tdf_bin and its contents.
I am not sure why the differences between the files are so stark: I can ask about it people from Bruker.

[Mnago]

1. No, it is a method for data conversion, it is an option in the acquisition sequence. I have checked the method and the screenshot of this method is attached.

2. OK, looking forward to your reply.

Best regards,

[Mnago]

Hi

1. I have confirmed that the 255387 ions extracted from MGF files were all precursor ions. So, the ions obtained from the TimsPy contains all the feature information.

2. It would be better that if we can get the charge state and intensity information from DDAPASEF and diaPASEF?

Best regards,

Winnie

[MatteoLacki]

Hello,

Ad 1. I am glad to hear that 👍

Ad 2. I am not sure about DDA_PASEF (I will check it), but regarding diaPASEF I am pretty sure that there is no such data available in the raw data. The raw data contains simply centroided peaks that need to be analyzed with a proper algorithm. Such algorithm might be part of the onboard software that is scheduling MS2 fragmentations based on particular peaks. In DIA one is fragmenting whole windows of precursors and I doubt that the onboard software could even cope with that amount of data.

Best wishes,

[Mnago]

Hi，

Yes. For the diaPASEF files, it is true that the charge state could not be labeled. The charge state information extracted from ddaPASEF files could be done by both DB Browser and MFG files. Hope it could be done by TimsPy.

How about the intensity of all features extracted from DDAPASEF files by timspy?