Dataset Information

Top-Down Mass Spectrometry of a Clinical Antibody Light Chain Using the Omnitrap-Orbitrap-Booster Platform

ABSTRACT: The Omnitrap-Orbitrap-Booster (OOB) mass spectrometry (MS) platform was developed to advance top-down (TD) MS analysis of proteins. It integrates a multimodal tandem mass spectrometry (MS/MS) ion trap system (OmnitrapTM), a high-resolution Orbitrap Fourier transform mass spectrometer (FTMS), and a high-performance data acquisition system (FTMS Booster) to improve fragmentation efficiency and spectral quality by increasing the signal-to-noise (S/N) ratio of product ions. In this study, we evaluate the OOB platform for electron capture dissociation (ECD)-based TD MS analysis of a clinical multiple myeloma antibody light chain (P15), benchmarking its performance against the “gold-standard” electron transfer dissociation (ETD)- based TD MS on an Orbitrap EclipseTM. Single precursor charge state analysis yielded comparable sequence coverage (68.2% vs. 74.3%) between ECD-based TD MS on OOB and ETD-based TD MS on EclipseTM. Notably, ECD exhibits lower spectral peak density (i.e., reduced spectral congestion) due to reduced redundancy of product ions. Furthermore, leveraging multiple precursor charge states (15+ to 19+) analysis consecutively across triplicate LC-MS/MS run on the OOB platform enhances sequence coverage to 93%, demonstrating its capacity for comprehensive protein characterization. These results establish the OOB platform as a powerful and efficient tool for TD MS of proteins.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Urine

DISEASE(S): Multiple Myeloma

SUBMITTER: Marie Yammine

LAB HEAD: Julia Chamot-Rooke

PROVIDER: PXD066881 | Pride | 2026-04-13

REPOSITORIES: Pride

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			Action	DRS
	20220712_QHF_TF_P15_LCs_red_alkyl_ECD50_120k_10us_AGC_fixed.raw	Raw
	20220712_QHF_TF_P15_LCs_red_alkyl_ECD50_120k_10us_AGC_fixed_1.raw	Raw
	20220712_QHF_TF_P15_LCs_red_alkyl_ECD50_120k_10us_AGC_prescan.raw	Raw
	20220712_QHF_TF_P15_LCs_red_alkyl_ECD50_120k_R1.raw	Raw
	20220712_QHF_TF_P15_LCs_red_alkyl_ECD50_120k_R2.raw	Raw

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Project description:Hardware changes introduced on the Orbitrap Ascend MultiOmics Tribrid MS include dual ion routing multipoles (IRMs) that can enable parallelized accumulation, dissociation, and Orbitrap mass analysis of three separate ion populations. The balance between these instrument functions is especially important in glycoproteomics, where complexities of glycopeptide fragmentation necessitate large precursor ion populations and, consequently, long ion accumulation times for quality MS/MS spectra. To compound matters further, dissociation methods like electron transfer dissociation (ETD) that benefit glycopeptide characterization come with overhead times that also slow down scan acquisition. Here we explored how the dual IRM architecture of the Orbitrap Ascend can improve glycopeptide analysis, with a focus on O-glycopeptide characterization using ETD with supplemental collisional activation (EThcD). We found that parallelization of ion accumulation and EThcD fragmentation – uniquely enabled by the Orbitrap Ascend – increased scan acquisition speed without sacrificing spectral quality, subsequently increasing the number of O-glycopeptides identified relative to analyses on the Orbitrap Eclipse (i.e., the previous generation Tribrid MS). Additionally, we systematically evaluated ion-ion reaction times and supplemental activation energies used for EThcD to understand how best to utilize acquisition time in the dual IRM architecture. We observed that shorter-than-expected ion-ion reaction times minimized scan overhead time without sacrificing c/z•-fragment ion generation, and that higher supplemental collision energies can generate combinations of glycan-retaining and glycan-neutral-loss peptide backbone fragments that benefit O-glycopeptide identification. We also saw improvements in N-glycopeptide analysis using collision-based dissociation, especially with methods using faster scan acquisition speeds. Overall, these data show how architectural changes to the Tribrid MS platform benefit glycoproteomic experiments by parallelizing scan functions to minimize overhead time and improve sensitivity.

Dataset Information

Top-Down Mass Spectrometry of a Clinical Antibody Light Chain Using the Omnitrap-Orbitrap-Booster Platform

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