DIA Method Validation

To establish the validity of the protein quantitation obtained from our 4 Da precursor mass window DIA workflow, we performed a dilution series of 72 SIL peptides in complex liver lysate using both 60 and 120 minute gradients. First, we looked at the number of points across the chromatographic peaks identified, a measure to determine the ability to accurately quantify a chromatographic peak. The 4 Da precursor mass windows can detect an average of 6.94 points across a peak in a 60 minute LC gradient while and the 120 minute LC gradient detected an average of 10.24 points across the peak. We next determined the accuracy of quantitation using both 60 and 120 minute gradients by quantifying the SIL peptides in our dilution series. We have linearity in quantification (r2 > 0.95) of  89/93 (95.7%) of transitions corresponding to precursors for the SIL peptides in the 60 gradient and linearity in quantification (r2 > 0.95) for 93/93 (100%) of the transitions corresponding to precursors for the SIL peptides in the 120 minute gradients.

 

Clustergrammer Heatmap
 
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60min_Gradient_SIL_Peptides_Dilution_Curve_DIA_2019-08-01_13-58-11.sky.zip2019-08-07 16:26:031701843,08821
120min_Gradient_SIL_Peptides_DIA_Dilution_Curve_2019-08-01_13-56-54.sky.zip2019-08-07 16:25:021701823,05221
120min_Gradient_SIL_Peptides_DIA_Dilution_Curve_12da_2019-08-01_13-55-57.sky.zip2019-08-07 16:24:021701823,0397