Here we present a parallel accumulation-mobility aligned fragmentation (PAMAF) mode that expands the capabilities of the MOBILion Systems MOBIE® high-resolution ion mobility (HRIM) system for mass spectrometry-based proteomics. The MOBIE system leverages structures for lossless ion manipulation (SLIM) to achieve HRIM separations within a compact footprint. Along with the HRIM separations, which increase the number of features identified per cycle, PAMAF enables alignment of fragment ions with their precursor ions without quadrupole isolation prior to fragmentation, avoiding the ion losses up to 99%. With PAMAF mode, ions can be accumulated in parallel to the ion mobility (IM) separation, achieving close to 100% ion utilization efficiency. Benchmarking results of PAMAF mode for a standard protein digest showed approximately 180x more protein group identifications for low-load proteomics workflows compared to standard data-dependent acquisition (DDA) analysis without HRIM.