Tips&Tricks - pFindStudio/pLink3 GitHub Wiki

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English

Running pLink via Command Line

Open a Command Prompt (cmd) Window
- Use the shortcut key win+R, then type cmd and press Enter
- Alternatively, you can google for how to open a cmd window
Navigate to the bin folder of the pLink installation directory
- Enter the command cd /d path and press Enter
  - Replace path with the actual path to the bin folder in your pLink installation directory
- For example: cd /d C:\pFindStudio\pLink\3.0.16\bin
Enter the command searcher.exe path_cfg.plink and press Enter
- Replace path_cfg.plink with the actual path to your search parameter configuration file, which should have a .plink extension
  - The .plink configuration file is generated by the pLink GUI and is located in the task results folder
  - The .plink file can be opened with a text editor, such as Notepad++
- Complete command example: searcher.exe F:\Temp\data_results\pLink_task_2025.03.06.04.13.28.plink
- For versions newer than pLink 3.0.16, multi-process mode is available
  - Multi-process command: MultiProcess.bat path_cfg.plink
  - We recommend using multi-process mode when there are many RAW files, as it will faster

Important Notes
- When running pLink via the command line, first ensure that spectral extraction has been completed in the GUI mode
  - Verify that the RAW file folder contains .pf2 files and _pParse2Plus.flag files
  - The number of .pf2 and _pParse2Plus.flag files should match the total number of RAW files
- We recommend always generate the .plink search parameter file via the GUI to avoid strange bugs
  - The contents of the parameter file may differ between different versions of pLink
Example Screenshot
- wiki_running_pLink_via_command_line_example

Extracting Spectra via Command Line

Open a Command Prompt (cmd) Window
- Use the shortcut win+R, then type cmd and press Enter
- Alternatively, you can google for how to open a cmd window
Navigate to the bin folder of the pLink installation directory
- Enter the command cd /d path and press Enter
  - Replace path with the actual path to the bin folder in your pLink installation directory
- For example: cd /d C:\pFindStudio\pLink\3.0.16\bin
Enter the command pParse2Plus.bat "path_cfg.plink" "param_pparse" "param_plus" and press Enter
- In the command, "path_cfg.plink" represents the actual path to your search parameter configuration file, which should have a .plink extension
  - The .plink file is generated by the pLink GUI and is located in the task results folder
  - The .plink file can be opened with a text editor, such as Notepad++
  - The .plink file records the path of each RAW file to be processed
    - Each line begins with spec_pathx =
    - Note: In spec_pathx = , x is the sequential number of the spectrum file (modify if necessary); spec_num = indicates the total number of RAW files (also remember to modify if needed)
    - The .raw suffix is changed to _HCDFT.pf2, indicating HCD fragmentation and Orbitrap acquisition data
    - For example:
```
spec_type = pf
spec_num = 3
spec_path1 = E:\dataset\xl\E1_HCDFT.pf2
spec_path2 = E:\dataset\xl\E2_HCDFT.pf2
spec_path3 = E:\dataset\xl\E3_HCDFT.pf2
```
  - Some other parameters in the .plink file are also used:
    - filter_tol = : MS1 matching tolerance, used for precursor deduplication
    - processor_num = : Number of processes, used for multi-process calling of pParse (default limited to max 12 processes considering disk I/O capability)
- In the command, "param_pparse" and "param_plus" represent the parameter strings for calling pParse and pParse2Plus respectively
  - When using the GUI, the first log line displays these parameters as reference, for example:
```
[pParse2Plus] "E:\xl\results\search\search.plink" " -t -0.8 -z 5 -i 1 -C 1 -a 0 -p 1 -S 1" " -path:pParse2 ..\pParse.exe -Plus:pxtract_n_isotope 2"
```
  - Important Note: For different MS Data Format, the parameters "param_pparse" and "param_plus" will differ.
    - For the differences between MS Data Format, please refer to: Choosing MS Data Format
- Complete command example, when the MS Data Format is RAW (Orbitrap):
```
pParse2Plus.bat "E:\xl\results\search\search.plink" " -t -0.8 -z 5 -i 1 -C 1 -a 0 -p 1 -S 1" " -path:pParse2 ..\pParse.exe -Plus:pxtract_n_isotope 2"
```

We still recommend: Use the GUI for spectrum extraction and pLink searches unless absolutely necessary
For running pLink via the command line, please refer to: Running pLink from the Command Line
If encounter any issues, please first refer to: Self-Troubleshooting for pLink3 Crashes or Prolonged Runtime

Searching CID-FT Data

CID-FT definition: Peptide fragmentation by CID, mass analysis using Orbitrap

Extract spectra using pLink interface (GUI)
- The pLink GUI does not support CID-FT data. After spectra extraction, the search will terminate.
- In the RAW file folder, you will find files ending with _CIDFT.pf2, which are binary-format spectral files extracted from RAW files.
- The number of .pf2 files should match the number of RAW files.
Manually modify the spectral file paths in the .plink search parameter file
- The .plink parameter file is generated by the pLink GUI and located in the task result folder.
- Open the .plink file with a text editor (e.g., Notepad++).
- Locate the parameter lines starting with spec_pathx = (where x is the sequential index of the spectral file).
  - Note: If multiple spectral files exist, ensure all relevant spec_pathx entries are modified.
  - Example:
```
[spectrum]
spec_title = HEK_db_2025.01.26
spec_type = pf
spec_num = 3
spec_path1 = E:\dataset\xl\E1_HCDFT.pf2
spec_path2 = E:\dataset\xl\E2_HCDFT.pf2
spec_path3 = E:\dataset\xl\E3_HCDFT.pf2
```
- Replace paths ending with _HCDFT.pf2 with your own _CIDFT.pf2 paths.
  - Example: spec_path1 = E:\dataset\xl\E1_CIDFT.pf2
Execute the search via command line
- For command-line usage, refer to: Running pLink via Command Line

Another Method
1. Similarly, wait for the pLink GUI to complete spectrum extraction (same as Step 1 in the previous method).
2. In the GUI:
  - Navigate to MS Data → Set MS Data Format to PF2.
  - Load the pre-extracted _CIDFT.pf2 spectral files.
3. Continue using the GUI to complete the parameter settings (without having to manually modify the .plink parameter file) and run the search.
Notes
- While this method enables pLink to search for CID-FT data, HCD fragmentation is still recommended as it generally provides more comprehensive fragmentation.
- pLink does not support low-resolution data (e.g., CID-IT, HCD-IT) acquired via Ion Trap (IT).

中文

命令行运行pLink

打开命令提示符（cmd）窗口
- 快捷键win+R, 然后输入cmd回车
- 或者你可自己谷歌，如何打开cmd窗口
进入到pLink安装目录的bin文件夹
- 输入命令cd /d path，然后回车
  - 命令中path表示pLink安装目录的bin文件夹路径
- 例如：cd /d C:\pFindStudio\pLink\3.0.16\bin
输入命令 searcher.exe path_cfg.plink，然后回车
- 命令中path_cfg.plink表示以 .plink为后缀的搜索参数文件路径
  - .plink文件由pLink界面生成，位置在任务结果文件夹
  - .plink文件可用文本编辑器打开，例如Notepad++
- 完整命令示例: searcher.exe F:\Temp\data_results\pLink_task_2025.03.06.04.13.28.plink
- 大于pLink3.0.16的版本有多进程模式
  - 多进程模式命令：MultiProcess.bat path_cfg.plink
  - 我们建议在RAW文件比较多时，使用多进程模式，速度会更快

注意
- 命令行运行pLink需要首先确认界面模式下，已经完成了谱图提取
  - 确认存放RAW的文件夹中，有产生 .pf2和 _pParse2Plus.flag结尾的文件。
    - _pParse2Plus.flag文件数目应与raw总数相同。
- .plink搜索参数文件，每次最好由界面生成，以避免奇怪的bug
  - 不同pLink版本间的参数文件内容可能会有不同
示例截图
- wiki_running_pLink_via_command_line_example

命令行提取谱图

打开命令提示符（cmd）窗口
- 快捷键 win+R, 然后输入 cmd回车
- 或者你可自己谷歌，如何打开cmd窗口
进入到pLink安装目录的bin文件夹
- 输入命令 cd /d path，然后回车
  - 命令中 path表示pLink安装目录的bin文件夹路径
- 例如：cd /d C:\pFindStudio\pLink\3.0.16\bin
输入命令pParse2Plus.bat "path_cfg.plink" "param_pparse" "param_plus"，然后回车
- 命令中"path_cfg.plink"表示以 .plink为后缀的搜索参数文件路径
  - .plink文件由pLink界面生成，位置在任务结果文件夹
  - .plink文件可用文本编辑器打开，例如Notepad++
  - .plink文件中记录了需要处理的每个RAW文件的路径
    - 每行以spec_pathx = 开始
    - 注意: spec_pathx = 中x是谱图文件的顺序编号，如有必要，不要忘记修改；spec_num =为RAW文件总数，也不要忘记修改
    - .raw后缀更改为了_HCDFT.pf2，意为是HCD碎裂，Orbitrap采集的数据
    - 例如：
```
spec_type = pf
spec_num = 3
spec_path1 = E:\dataset\xl\E1_HCDFT.pf2
spec_path2 = E:\dataset\xl\E2_HCDFT.pf2
spec_path3 = E:\dataset\xl\E3_HCDFT.pf2
```
  - .plink中部分其它参数也被使用
    - filter_tol = ：MS1匹配误差，用于Precursors去冗余
    - processor_num = : 进程数，用于多进程调用pParse（考虑到硬盘读写能力，默认限制最高12进程）
- 命令中"param_pparse"、"param_plus"分别表示调用pParse和pParse2Plus的参数字符串
  - 在使用界面时，第一行日志会显示该参数，可作为参考，例如：
```
[pParse2Plus] "E:\xl\results\search\search.plink" " -t -0.8 -z 5 -i 1 -C 1 -a 0 -p 1 -S 1" " -path:pParse2 ..\pParse.exe -Plus:pxtract_n_isotope 2"
```
  - 注意: 不同的MS Data Format，"param_pparse"和"param_plus"参数会不同
  - 不同MS Data Format区别，请参考：选择MS Data Format
- 完整命令示例，当MS Data Format为RAW (Orbitrap)时：
```
pParse2Plus.bat "E:\xl\results\search\search.plink" " -t -0.8 -z 5 -i 1 -C 1 -a 0 -p 1 -S 1" " -path:pParse2 ..\pParse.exe -Plus:pxtract_n_isotope 2"
```

我们仍然建议：在不必须的情况下，使用界面提取谱图和运行pLink搜索
命令行运行pLink，可参考：命令行运行pLink
遇到问题，可先参考：pLink3运行崩溃或时间过长-自我排查

搜索CID-FT数据

CID-FT指代：肽段碎裂方式为CID，质量分析使用Orbitrap

使用pLink界面提取谱图
- 因pLink界面不支持CID-FT数据，所以等界面提取完谱图后，搜索会终止
- 在存放RAW的文件夹中，会发现以_CIDFT.pf2结尾的文件，这即是从RAW提取的谱图的二进制格式文件
- .pf2的文件数目应该与RAW文件数一样
手动修改.plink搜索参数文件中的谱图文件路径
- .plink搜索参数文件由pLink界面生成，位置在任务结果文件夹
- .plink文件可用文本编辑器打开，例如Notepad++
- 谱图文件路径的参数的行，以spec_pathx = 开始
  - 注意: spec_pathx = 中x是谱图文件的顺序编号，如有必要，不要忘记修改
  - 例如:
```
[spectrum]
spec_title = HEK_db_2025.01.26
spec_type = pf
spec_num = 3
spec_path1 = E:\dataset\xl\E1_HCDFT.pf2
spec_path2 = E:\dataset\xl\E2_HCDFT.pf2
spec_path3 = E:\dataset\xl\E3_HCDFT.pf2
```
- 将_HCDFT.pf2为结尾的路径，修改为你自己的_CIDFT.pf2的路径
  - 例如: spec_path1 = E:\dataset\xl\E1_CIDFT.pf2
使用命令行运行搜索参数文件
- 命令行模式教程：命令行运行pLink

另外一种方法
1. 同样，等待pLink界面提取谱图完成，和上面方法第1步相同
2. 使用界面
  - MS Data → MS Data Format选择PF2
  - 载入已经提取完的_CIDFT.pf2谱图文件
3. 继续使用界面完成参数设置后（不用再手动修改.plink参数文件），进行搜索
注意
- 虽然通过这种方法可以使得pLink搜索CID-FT数据，但我们仍然建议使用HCD进行肽碎裂，一般HCD碎裂相对更充分
- 现在pLink不支持CID-IT、HCD-IT等低精度Ion Trap（IT）采集的数据