Dataset Information

Understanding m/z range settings for MS/MS scans: a case study with intact glycopeptides

ABSTRACT: Several practical considerations dictate the scan range, or breadth of ion m/z values, that can be transmitted through ion guides in mass spectrometers (MS). Voltage settings must be balanced to minimize low m/z cutoffs while also generating effective pseudopotential wells for high m/z ions to maximize transmission of ion populations of interest. The scan range is particularly impactful in glycoproteomics, because glycopeptide identification typically relies on low mass-to-charge (m/z) glycan-derived oxonium ions and higher m/z peptide fragments that retain glycan modifications. Common practice suggests a so-called “5-10-15 rule” when setting scan ranges, which defines the upper m/z value to be used for the scan based on a multiple of the first m/z value. While this axiom has very real implications for transmitting diverse ion populations in full MS scans, adhering to this strategy can unnecessarily limit analytical information available in glycopeptide MS/MS spectra. Here, we explore the implications of following or breaking the 5-10-15 rule in MS/MS scans for glycopeptide characterization on a quadrupole-Orbitrap-linear ion trap Tribrid MS system (Orbitrap Ascend). We show that breaking the 5-10-15 rule does not lead to a significant loss of fragment ion transmission at either extreme of the m/z range. We demonstrate this concept for glycopeptides fragmented with higher-energy collisional dissociation (HCD), electron-transfer dissociation (ETD), and electron-transfer/higher-energy collision dissociation (EThcD). We use this case study to discuss the concepts important to using the 5-10-15 rule wisely and when it can be practically ignored, such as using large m/z ranges to improve glycopeptide characterization.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Blood Cell

SUBMITTER: Tim S Veth

LAB HEAD: Nicholas M. Riley

PROVIDER: PXD062360 | Pride | 2026-06-08

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	240110_TVKK_R00018_MG_c00001_glyco_ETD.raw	Raw
	240110_TVKK_R00018_MG_c00001_glyco_EThcD.raw	Raw
	240124_TVKK_R00018_MG_c00001_SA_120-1200.raw	Raw
	240124_TVKK_R00018_MG_c00001_SA_glyco_HCD.raw	Raw
	File_S1_240124_TVKK_R00018_MG_c00001_SA_glyco_HCD_Spectra.pdf	Pdf

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Publications

Understanding m/z Range Settings for MS/MS Scans: A Case Study with Intact Glycopeptides.

Veth Tim S TS Kothlow Kathryn K Riley Nicholas M NM

Journal of the American Society for Mass Spectrometry 20260223 4

Effective glycopeptide identification with tandem mass spectrometry (MS/MS) often relies on both low mass-to-charge (m/z) ions derived from glycan-specific oxonium ions and higher m/z peptide fragment ions that retain glycan modifications. Thus, glycoproteomic experiments benefit from a wider MS/MS scan range, i.e., the breadth of m/z values measured in fragmentation spectra, than those typically used in nonmodified peptide analyses. Here, we explore the ...[more]

PMID: 41731685

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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.

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.

Dataset Information

Understanding m/z range settings for MS/MS scans: a case study with intact glycopeptides

Dataset's files

Publications

Understanding <i>m</i>/<i>z</i> Range Settings for MS/MS Scans: A Case Study with Intact Glycopeptides.

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OmicsDI is part of the ELIXIR infrastructure

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Dataset Information

Understanding m/z range settings for MS/MS scans: a case study with intact glycopeptides

Dataset's files

Publications

Understanding &lt;i&gt;m&lt;/i&gt;/&lt;i&gt;z&lt;/i&gt; Range Settings for MS/MS Scans: A Case Study with Intact Glycopeptides.

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OmicsDI is part of the ELIXIR infrastructure

Tweets

Understanding <i>m</i>/<i>z</i> Range Settings for MS/MS Scans: A Case Study with Intact Glycopeptides.