Estimating the Reliability of Low-Abundant Signals and Limited Replicate Measurements through MS2 Peak Area in SWATH.

Sequential windows acquisition of all theoretical fragment ions mass spectrometry (SWATH-MS) provides large-scale protein quantification with high accuracy and selectivity. Nevertheless, reliable quantification of low-abundant signals in complex samples remains challenging, as recently illustrated in a multicenter benchmark study of different label-free software tools. Here, the SWATH Replicates Analysis 2.0 template from Sciex is used to highlight that the relationship between the MS2 peak area and the variability can be described by a function. This functional relationship appears to be largely insensitive to variation in samples or acquisition conditions, suggesting a device-intrinsic property. By using a power regression, it is shown that the MS2 peak area can be used to predict the quantification repeatability without relying on replicate injections, thus contributing to high-throughput confident quantification of low-abundant signals with SWATH-MS.