Bottom-Up Quantitative Proteomics Workflows for Parallel Accumulation Mobility Aligned Fragmentation (PAMAF) Analysis

Parallel Accumulation Mobility Aligned Fragmentation (PAMAFTM) analysis is a novel data independent acquisition mode that uses high-resolution ion mobility (HRIM) separation to align precursor and fragment ions without quadrupole isolation. Here, we evaluate PAMAF performance for high-throughput protein quantitation.
Data were acquired using a prototype SLIM-based MOBIE HRIM system coupled to an Agilent 6545 QTOF mass spectrometer. Quantitative standards (Pierce Retention Time Calibration and MS Qual/Quant QC Mix) and heavy labeled peptides (SpikeMixTM Cytokines) were used to evaluate PAMAF mode quantitative abilities. Relative abundances of modified species were evaluated using HeLa and NISTmAb tryptic digest standards. Data were processed in Skyline and Spectronaut v19, with additional ion mobility visualization in IM-MS Browser.
PAMAF analysis demonstrated improved MS1 and MS2 signal intensity and increased fragment ion coverage relative to DDA. HRIM separation reduced chimeric spectra from coeluting, isobaric peptides and improved spectral match scores, yielding over a threefold increase in protein identifications relative to DDA benchmarks, including several low-level modifications uniquely identified in the PAMAF analysis.
Quantitative robustness was demonstrated at high speeds in HeLa spike d with SpikeMix peptides experiments, where over 85% of 461 peptides at 200 fmol were quantifiable at 60 samples/day and over 65% remained quantifiable at 500 samples/day. At lower abundance (2 fmol), identification rates remained stable across the same throughput range. Short MS1/MS2 cycle times (400–800 ms) increased data point density across chromatographic peaks, supporting precise quantitation under fast LC conditions.
PAMAF mode combines HRIM-enabled ion utilization with ultra-high MS/MS rates to deliver sensitive, precise, and scalable protein quantitation, positioning HRIM-QTOF platforms as a powerful solution for high-throughput quantitative proteomics.