Trapped Ion Mobility Spectrometry Reduces Spectral Complexity in Mass Spectrometry Based Workflow

In bottom-up mass spectrometry based proteomics, deep proteome coverage is limited by high cofragmentation rates. This occurs when more than one analyte is isolated by the quadrupole and the subsequent fragmentation event produces fragment ions of heterogeneous origin. One strategy to reduce cofragmentation rates is through effective peptide separation techniques such as chromatographic separation and, the more recently popularized, ion mobility (IM) spectrometry which separates peptides by their collisional cross section. Here we investigate the capability of the Trapped Ion Mobility Spectrometry (TIMS) device to effectively separate peptide ions and quantify the separation power of the TIMS device in the context of a Parallel Accumulation-Serial Fragmentation (PASEF) workflow. We found that TIMS IM separation increases the number of interference-free MS1 features 9.2-fold, while decreasing the average peptide density in precursor spectra 6.5 fold. In a Data Dependent Acquisition (DDA) PASEF workflow, IM separation increased the number of spectra without cofragmentation by a factor of 4.1 and the number of high quality spectra 17-fold. This observed decrease in spectral complexity results in a substantial increase in peptide identification rates when using our data-driven model. In the context of a Data Independent Acquisition (DIA), the reduction in spectral complexity resulting from IM separation is estimated to be equivalent to a 4-fold decrease in isolation window width (from 25Da to 6.5Da). Our study shows that TIMS IM separation dramatically reduces cofragmentation rates leading to an increase in peptide identification rates.