Work

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

Year: 2026

Type: article

Abstract: 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 implications of breaking a common axiom for scan range settings called the “5–10–15 rule” used to maximize transmission of ion populations of interest. This 5–10–15 rule, which defines the upper m / z value for a scan as a multiple of the first m / z value, comes from fundamental requirements for stable ion trajectories, where voltage settings must balance retention of low m / z ions while also generating effective pseudopotential wells to trap high m / z ions. Adhering to this calculation for MS/MS scan range settings can reduce glycopeptide ion coverage by excluding the analysis of either low m / z oxonium ions or high m / z fragment ions. We use a quadrupole-Orbitrap-linear ion trap Tribrid MS system (Orbitrap Ascend) to investigate the implications of following or breaking the 5–10–15 rule in MS/MS scans for glycopeptide characterization with higher-energy collisional dissociation (HCD), electron-transfer dissociation (ETD), and electron-transfer/higher-energy collision dissociation (EThcD). For scans with a first m / z value around m / z 120 (i.e., capturing most common glycan-specific oxonium ions), 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 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 scan ranges to improve glycopeptide characterization.

Source: Journal of the American Society for Mass Spectrometry

DOI: 10.1021/jasms.5c00352

Authors: Tim S. Veth, Kathryn Kothlow, Nicholas M. Riley

Institution: University of Washington

Language: English

Topic: Mass Spectrometry Techniques and Applications

Subfield: Spectroscopy

Field: Chemistry

Domain: Physical Sciences

Open Access status: green

Funders: Kinship Foundation, National Institutes of Health, National Institute of General Medical Sciences, American Society for Mass Spectrometry

Awards: G3418984644, Molecular Biophysics Training Program, National Center for Quantitative Biology of Complex Systems, Capturing the Holistic Glycocode through Systems Glycobiology