Openswath error due to low rsq when running short-gradient DIA MS runs

[vidyavenkatraman]

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.