Dataset Information

Improvements in glycoproteomics through architecture changes to the Orbitrap Tribrid MS platform

ABSTRACT: 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.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Tim S Veth

LAB HEAD: Nicholas M. Riley

PROVIDER: PXD058153 | Pride | 2025-09-14

REPOSITORIES: Pride

ACCESS DATA

Dataset's files

Source:

			Action	DRS
	22-12-14_EclipseOglyco_EThcD_100ms_30msRxn_1.raw	Raw
	22-12-14_EclipseOglyco_EThcD_100ms_30msRxn_2.raw	Raw
	22-12-14_EclipseOglyco_EThcD_100ms_50msRxn_1.raw	Raw
	22-12-14_EclipseOglyco_EThcD_100ms_50msRxn_2.raw	Raw
	22-12-14_EclipseOglyco_EThcD_100ms_70msRxn_1.raw	Raw

Items per page:

1 - 5 of 226

Publications

Improvements in Glycoproteomics through Architecture Changes to the Orbitrap Tribrid MS Platform.

Veth Tim S TS Sutherland Emmajay E Markuson Kayla A KA Zhang Ruby R Duboff Anna G AG Huang Jingjing J Bergen David D Lee Amanda E AE Melani Rafael D RD Canterbury Jesse D JD Zabrouskov Vlad V McAlister Graeme C GC Mullen Christopher C Riley Nicholas M NM

Analytical chemistry 20250529 22

Hardware changes introduced on the Orbitrap Ascend Tribrid MS include dual ion routing multipoles (IRMs) that 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 long ion accumulation times for quality MS/MS spectra. To compound matters further, dissocia ...[more]

PMID: 40439173

Similar Datasets

Project description:Isolated murine kidney glomeruli were lysed in buffer containing 8M urea, and proteins were reduced and alkylated. Proteins were digested with trypsin. After reverse-phase chomatrography, peptides were fractionated using strong cation exchange chromatography. Phosphopeptides were enriched using IMAC (FeNTA) columns. Peptides were analyzed by LC-MS/MS on a Q Exactive mass spectrometer or an LTQ Orbitrap XL mass spectrometer. Metadata for Orbitrap samples (MaxQuant Output files include „Orbi“) 1. General features – 1.1 Global descriptors a. Responsible person or role: Markus Rinschen. markus.rinschen@uk-koeln.de; tobias.lamkemeyer@uni-koeln.de b. Instrument manufacturer, model: LTQ-Orbitrap XL, Thermo Scientific c. customization 2. Ion sources – ESI fed by nLC 2 (Proxeon) 3. Post source component 3.1. Analyser: MS1 survey scan in an Orbitrap and MS2 analysed in Linear Trap. 3.2. Activation/Dissociation: CID 4. Spectrum and peak list generation and annotation 4.1. Data acquisition: MaxQuant v. 1.3.05 Top one method with a cycle of one full MS1 scan in the Orbitrap, followed by the fragmentation with dynamic exclusion window of 60 seconds and followed by the acquisition of 5 product ion scans generated in the LTQ analyser, and detected in the LTQ. Unselected fragmentation. For further details, see .raw files. URL of file: Filename 4.2. Data analysis: MaxQuant v 1.3.05 Parameters used in the generation of peak lists or processed spectra: see annotation in “parameters” file. The mouse uniprot reference database “complete proteome” obtained on January 2nd, 2013 was used. Metadata for QExactive samples (Max Quant output files include „QE“) 1. General features – 1.1 Global descriptors a. Responsible person or role: Markus Rinschen. markus.rinschen@uk-koeln.de; Marcus Krüger marcus.krueger@mpi-bn.mpg.de b. Instrument manufacturer, model: Q Exactive Thermo Scientific c. customization 2. Ion sources – ESI fed by nLC 1000 (Proxeon) 3. Post source component 3.1. Analyser: MS1 survey scan in an Orbitrap and MS2 analysed in Orbitrap/HCD cell 3.2. Activation/Dissociation: HCD 4. Spectrum and peak list generation and annotation 4.1. Data acquisition: MaxQuant v. 1.3.05 Top one method with a cycle of one full MS1 scan in the Orbitrap, followed by the fragmentation with dynamic exclusion window of 20 seconds in the HCD cell and followed by the acquisition of 10 product ion scans generated in the LTQ analyser, and detected in the LTQ. Unselected fragmentation. For further details, see .raw files. URL of file: Filename 4.2. Data analysis: MaxQuant v 1.3.05 Parameters used in the generation of peak lists or processed spectra: see annotation in “parameters” file. The mouse uniprot reference database “complete proteome” obtained on January 2nd, 2013 was used.