A Parallel Accumulation-Mobility Aligned Fragmentation Strategy Utilizing High-Resolution Ion Mobility for...

Area of Science:

• Mass Spectrometry
• Proteomics
• Analytical Chemistry

Background:

• Data independent acquisition (DIA) is crucial for proteomics discovery workflows.
• Traditional quadrupole filtering in MS can be slow and lead to ion loss.
• High-resolution ion mobility (HRIM) offers advanced separation capabilities.

Purpose of the Study:

• Introduce a novel DIA-MS operating mode, parallel accumulation-mobility aligned fragmentation (PAMAF).
• Leverage HRIM and structures for lossless ion manipulation (SLIM) for enhanced precursor isolation.
• Improve speed, sensitivity, and identification accuracy in bottom-up proteomics.

Main Methods:

• Developed and implemented PAMAF mode using HRIM for precursor isolation, replacing quadrupole filtering.
• Utilized mobility-based time alignment to associate fragment ions with precursors.
• Achieved ~100% ion utilization efficiency through ion accumulation during analysis.

Main Results:

• LC-PAMAF-MS identified ~6x more protein groups than standard data-dependent acquisition (DDA) in whole cell digests.
• Achieved >100x improvement for low-load workflows and quantified low-abundance peptides undetectable by DDA.
• Resolved coeluting isobars and isomers prior to fragmentation, eliminating chimeric spectra.

Conclusions:

• PAMAF mode significantly enhances speed and sensitivity in MS-based proteomics discovery.
• The technique improves protein identification, quantification, and spectral quality.
• DIA-PAMAF mode, combining HRIM and quadrupole isolation, detected over 8,000 protein groups in HeLa digests, showcasing improved specificity.