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 precursors 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. Quantitative evaluations demonstrated that PAMAF mode was capable of quantifying low abundance peptides, including those undetectable by DDA.