A Parallel Accumulation-Mobility Aligned Fragmentation Strategy Utilizing High-Resolution Ion Mobility for High Performance Proteomics Analysis

ABSTRACT Here we present a novel data independent acquisition (DIA) mass spectrometry (MS) operating mode termed parallel accumulation-mobility aligned fragmentation (PAMAF) that offers enhanced speed and sensitivity of ion fragmentation analysis for nontargeted discovery workflows such as bottom-up proteomics. This mode of operation leverages high-resolution ion mobility (HRIM) separation capabilities of the structures for lossless ion manipulation (SLIM) technology to achieve HRIM-based precursor isolation in place of traditional quadrupole filtering approaches. This PAMAF mode of operation increases the number of features that can be identified per MS1/MS2 acquisition cycle by employing mobility-based time alignment to associate fragment ions with their corresponding precursor ions. By using a high-speed, lossless separation technique for precursor isolation instead of the comparatively slow and wasteful quadrupole filtering method, we can avoid ion losses up to 99% while simultaneously increasing the rate at which precursor ions are sequentially fragmented and detected. Additionally, by storing ions in a trapping region while the previous packet of ions is being analyzed, the PAMAF mode achieves ∼100% ion utilization efficiency. Benchmarking results of LC-PAMAF-MS analysis of a whole cell protein digest showed approximately 6x more protein group identifications compared to a standard data-dependent acquisition (DDA) analysis without HRIM on the same QTOF instrument, and to over 100x improvement for low-load workflows. Quantitative evaluations demonstrated that PAMAF mode could quantify low abundance peptides, including those undetectable by DDA. Additionally, since precursor isolation in PAMAF mode is size-based rather than m/z -based, many coeluting isobars and isomers can be resolved prior to fragmentation to eliminate chimeric spectra that compromise identification accuracy. In this work we also explored the benefits of combining HRIM and quadrupole isolation to achieve improved specificity. This approach, known as DIA-PAMAF mode, further reduces the frequency of chimeric fragmentation spectra, and enabled the detection of over 8,000 protein groups from a HeLa digest analysis. PAMAF mode brings a powerful new technique to the field of proteomics that has the potential to improve the sensitivity and selectivity of mass spectrometry-based proteomics.