ABSTRACT Data-independent acquisition (DIA) methods have become increasingly attractive in mass spectrometry (MS)-based proteomics, because they enable high data completeness and a wide dynamic range. Recently, we combined DIA with parallel accumulation – serial fragmentation (dia-PASEF) on a Bruker trapped ion mobility separated (TIMS) quadrupole time-of-flight (TOF) mass spectrometer. This requires alignment of the ion mobility separation with the downstream mass selective quadrupole, leading to a more complex scheme for dia-PASEF window placement compared to DIA. To achieve high data completeness and deep proteome coverage, here we employ variable isolation windows that are placed optimally depending on precursor density in the m/z and ion mobility plane. This A utomatic Isolation D esign procedure is implemented in the freely available py_diAID package. In combination with in-depth project-specific proteomics libraries and the Evosep LC system, we reproducibly identified over 7,700 proteins in a human cancer cell line in 44 minutes with quadruplicate single-shot injections at high sensitivity. Even at a throughput of 100 samples per day (11 minutes LC gradients), we consistently quantified more than 6,000 proteins in mammalian cell lysates by injecting four replicates. We found that optimal dia-PASEF window placement facilitates in-depth phosphoproteomics with very high sensitivity, quantifying more than 35,000 phosphosites in a human cancer cell line stimulated with an epidermal growth factor (EGF) in triplicate 21 minutes runs. This covers a substantial part of the regulated phosphoproteome with high sensitivity, opening up for extensive systems-biological studies.

Rapid and in-depth coverage of the (phospho-)proteome with deep libraries and optimal window design for dia-PASEF