A Dual-Gated Structures for Lossless Ion Manipulations-Ion Mobility Orbitrap Mass Spectrometry Platform for Combined Ultra-High-Resolution Molecular Analysis

High-resolution ion mobility spectrometry-mass spectrometry(HR-IMS-MS)instruments have enormously advanced the ability to characterize complexbiological mixtures. Unfortunately, HR-IMS and HR-MS measurementsare typically performed independently due to mismatches in analysistime scales. Here, we overcome this limitation by using a dual-gatedion injection approach to couple an 11 m path length structures forlossless ion manipulations (SLIM) module to a Q-Exactive Plus OrbitrapMS platform. The dual-gate setup was implemented by placing one iongate before the SLIM module and a second ion gate after the module.The dual-gated ion injection approach allowed the new SLIM-Orbitrapplatform to simultaneously perform an 11 m SLIM separation, Orbitrapmass analysis using the highest selectable mass resolution setting(up to 140 k), and high-energy collision-induced dissociation (HCD)in similar to 25 min over an m/z range of similar to 1500amu. The SLIM-Orbitrap platform was initially characterized usinga mixture of standard phosphazene cations and demonstrated an averageSLIM CCS resolving power (Rp(CCS)) of similar to 218 and anSLIM peak capacity of similar to 156, while simultaneously obtaininghigh mass resolutions. SLIM-Orbitrap analysis with fragmentation wasthen performed on mixtures of standard peptides and two reverse peptides(SDGRG(1+), GRGDS(1+), and Rp(CCS) = 305)to demonstrate the utility of combined HR-IMS-MS/MS measurements forpeptide identification. Our new HR-IMS-MS/MS capability was furtherdemonstrated by analyzing a complex lipid mixture and showcasing SLIMseparations on isobaric lipids. This new SLIM-Orbitrap platform demonstratesa critical new capability for proteomics and lipidomics applications,and the high-resolution multimodal data obtained using this systemestablish the foundation for reference-free identification of unknownion structures.

Automated summary

High-resolution ion mobility spectrometry-mass spectrometry (HR-IMS-MS) instruments have significantly enhanced the characterization of complex biological mixtures. By using a dual-gated ion injection approach, we coupled an 11 m path length SLIM module to a Q-Exactive Plus Orbitrap MS platform, enabling simultaneous SLIM separation, high-resolution Orbitrap mass analysis, and high-energy collision-induced dissociation (HCD). The SLIM-Orbitrap platform was characterized using standard phosphazene cations, standard peptides, and complex lipid mixtures, demonstrating its capability for peptide identification and isobaric lipid separations.