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

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.