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

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 on ion trapping-based instruments 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 for MS/MS scans 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.