High sensitivity dia-PASEF proteomics with DIA-NN and FragPipe

The dia-PASEF technology exploits ion mobility separation for high-sensitivity analysis of complex proteomes. Here, we demonstrate neural network-based processing of the ion mobility data, which we implement in the DIA-NN software suite. Using spectral libraries generated with the MSFragger-based FragPipe computational platform, the DIA-NN analysis of dia-PASEF raw data increases the proteomic depth by up to 69% compared to the originally published dia-PASEF workflow. For example, we quantify over 5200 proteins from 10ng of HeLa peptides separated with a 95-minute nanoflow gradient, and over 5000 proteins from 200ng using a 4.8-minute separation with an Evosep One system. In complex samples, featuring a mix of human and yeast lysates, the workflow detects over 11700 proteins in single runs acquired with a 100-minute nanoflow gradient, while demonstrating quantitative precision. Hence, the combination of FragPipe and DIA-NN provides a simple-to-use software platform for dia-PASEF data analysis, yielding significant gains in high-sensitivity proteomics.