Dear Skyline Team,

1. I have just started working with SWATH analysis. I wanted to check the biological variation in my system. Hence, I isolated proteins from the roots of 6 plants of the same cultivar and age and subjected them to DIA (80 variable window, TTOF 6600). I am using a spectral library prepared from the DDA runs of the same sample pools injected 6 times. Please let me know if I can use the peak area histograms as an indicator of the biological variation. I am getting a median of 19.4% (Below 20%: 52.3%) for the products and for the precursors it is 33.5% (Below 20%: 28.2%). Which one should I consider? Does it mean that only 28.2% of my proteins can be reliably quantified at the protein level?

2. I also did another experiment where I isolated proteins from 3 healthy and 3 diseased roots as a pilot study and did both a DDA and DIA analysis, as above. Control MSstats as an external tool: I could successfully install the tool but I am not able to do the group comparison using MSstats (Skyline's group comparison works). What should be my MSstats input settings in Skyline? Should I use MS1 or MS2 for group comparison? I get the following error.

""C:\Program Files\R\R-3.5.0\bin\R.exe" -f "C:\Users\u15407218\AppData\Local\Apps\2.0\QJGPLL6E.O5C\LTQ26TN4.44Z\skyl..tion_e4141a2a22107248_0004.0001_8a31a35b7206059e\Tools\MSstats-3.13.6\MSStatsGC.r" --slave --args "C:\Users\u15407218\AppData\Local\Temp\MSstats_Group_Comparison_MSstats_Input.csv" ControlvsInfected 1 FALSE FALSE FALSE -1 1
  ================================================================

 ** Loading the required statistical software packages in R .....

  =======================================

 ** Reading the data for MSstats.....

** iRT proteins/peptides are removed.

** Peptides, that are used in more than one proteins, are removed.

** Truncated peaks are replaced with NA.

Error in SkylinetoMSstatsFormat(raw, removeProtein_with1Feature = TRUE, :

  ** Please check precursors information. If your experiment is DIA, please remove the precursors. If your experiments is DDA, please check the precursor information.

  Can't finish analysis."


Regarding determination of sample size, I tried the analysis using Skyline external tool option and once again I get an error message. What should be my MSstats input settings in Skyline? Following error:

""C:\Program Files\R\R-3.5.0\bin\R.exe" -f "C:\Users\u15407218\AppData\Local\Apps\2.0\QJGPLL6E.O5C\LTQ26TN4.44Z\skyl..tion_e4141a2a22107248_0004.0001_8a31a35b7206059e\Tools\MSstats-3.13.6\MSStatsDSS.r" --slave --args "C:\Users\u15407218\AppData\Local\Temp\MSstats_Design_Sample_Size_MSstats_Input.csv" 1 TRUE 0.80 0.05 1.25 1.75 FALSE FALSE FALSE
================================================================
 ** Loading the required statistical software packages in R .....
  =======================================
 ** Reading the data for MSstats.....
** Peptides, that are used in more than one proteins, are removed.
Error in `$<-.data.frame`(`*tmp*`, "Intensity", value = numeric(0)) :
  replacement has 0 rows, data has 129474

 Can't finish analysis."

--
Thanking you,
Best regards,
Ashok