We are trying a new method using Thermo Exploris to acquire DIA-MS plasma runs with 21Da windows on a 5 minute gradient. When I tried to analyze this data using openswathworkflow against the Twin library, it seems to be having a hard time performing RT normalization and fails with a low rsq - see error below:
Progress of 'Load TraML file':
-- done [took 0.02 s (CPU), 0.03 s (Wall)] --
Progress of 'Extract iRT chromatograms':
-- done [took 0.14 s (CPU), 0.14 s (Wall)] --
Progress of 'Retention time normalization':
Will analyse 10 peptides with a total of 56 transitions
rsd < 0.0
Intercept 117.811
Slope -0.504536
Squared pearson coefficient 0.292509
Value of the t-distribution 2.57058
Standard deviation of the residuals 13.8761
Standard error of the slope 0.139571
The X intercept 233.503
The lower border of confidence interval 202.136
The higher border of confidence interval 376.309
Chi squared value 6085.67
x mean 179.034
stand_error_slope/slope_ -27.5026
Coefficient of Variation -15.3617
=========================================
rsq: 0.292509 points: 7
rsd < 0.0
Intercept 117.811
Slope -0.504536
Squared pearson coefficient 0.292509
Value of the t-distribution 2.57058
Standard deviation of the residuals 13.8761
Standard error of the slope 0.139571
The X intercept 233.503
The lower border of confidence interval 202.136
The higher border of confidence interval 376.309
Chi squared value 6085.67
x mean 179.034
stand_error_slope/slope_ -27.5026
Coefficient of Variation -15.3617
=========================================
rsd < 0.0
Intercept 81.7185
Slope -0.349548
Squared pearson coefficient 0.246127
Value of the t-distribution 2.77645
Standard deviation of the residuals 10.6092
Standard error of the slope 0.111326
The X intercept 233.783
The lower border of confidence interval 197.264
The higher border of confidence interval 631.554
Chi squared value 3398.77
x mean 183.405
stand_error_slope/slope_ -30.3511
Coefficient of Variation -16.5487
=========================================
rsq: 0.246127 points: 6
rsd < 0.0
Intercept 81.7185
Slope -0.349548
Squared pearson coefficient 0.246127
Value of the t-distribution 2.77645
Standard deviation of the residuals 10.6092
Standard error of the slope 0.111326
The X intercept 233.783
The lower border of confidence interval 197.264
The higher border of confidence interval 631.554
Chi squared value 3398.77
x mean 183.405
stand_error_slope/slope_ -30.3511
Coefficient of Variation -16.5487
=========================================
rsd < 0.0
Intercept 67.8974
Slope -0.229684
Squared pearson coefficient 0.226375
Value of the t-distribution 3.18245
Standard deviation of the residuals 6.01437
Standard error of the slope 0.0652778
The X intercept 295.612
The lower border of confidence interval 233.667
The higher border of confidence interval 1406.64
Chi squared value 1530.43
x mean 178.96
stand_error_slope/slope_ -26.1854
Coefficient of Variation -14.632
=========================================
rsq: 0.226375 points: 5
rsd < 0.0
Intercept 67.8974
Slope -0.229684
Squared pearson coefficient 0.226375
Value of the t-distribution 3.18245
Standard deviation of the residuals 6.01437
Standard error of the slope 0.0652778
The X intercept 295.612
The lower border of confidence interval 233.667
The higher border of confidence interval 1406.64
Chi squared value 1530.43
x mean 178.96
stand_error_slope/slope_ -26.1854
Coefficient of Variation -14.632
=========================================
Error: Unexpected internal error (WARNING: rsq: 0.226374750572749 is below limit of 0.94999999999999996. Validate assays for RT-peptides and adjust the limit for rsq or coverage.)
I was able to check that all the iRT peptides were detected using QuiC and Skyline.
.
Any advice on why the Rsq normalization would fail on very short gradients (5 minute) and if there are any parameters i can try to adjust to proceed further would be highly appreciated.
We are trying a new method using Thermo Exploris to acquire DIA-MS plasma runs with 21Da windows on a 5 minute gradient. When I tried to analyze this data using openswathworkflow against the Twin library, it seems to be having a hard time performing RT normalization and fails with a low rsq - see error below:
I was able to check that all the iRT peptides were detected using QuiC and Skyline.
.
Any advice on why the Rsq normalization would fail on very short gradients (5 minute) and if there are any parameters i can try to adjust to proceed further would be highly appreciated.