Targeted N-Glycan Analysis with Parallel Reaction Monitoring Using a Quadrupole-Orbitrap Hybrid Mass Spectrometer

Abstract

Targeted mass spectrometric analysis is widely employed across various omics fields. The approach has been successfully employed for the structural analysis of proteins, glycans, lipids, and small molecules. Selected reaction monitoring and multiple reaction monitoring (MRM) have been a method of choice for targeted structural studies of biomolecules. However, innovations in instrument designs have led to the development of parallel reaction monitoring (PRM). PRM detects all product ions simultaneously rather than optimizing/preselecting the target glycan transitions, simplifying the analytical workflow. By reducing background interference, increasing selectivity/specificity, and improving data quality, PRM allows reliable quantification of target glycans in complex matrices. PRM can also improve sensitivity for detecting low-abundance target glycans and reduce low-level limit of quantification values with an improved S/N ratio. PRM’s advantages are attributed to the development of sensitive and highly selective mass analyzers, orbitrap, and time of flight. In this study, we developed a sensitive PRM method for the quantitative analysis of permethylated N-glycans, an important class of disease biomarkers, using a quadrupole-orbitrap hybrid mass spectrometer. Pooled human cerebrospinal fluid was used for the study as a source of permethylated N-glycans. The method illustrates the fragmentation of N-glycans at different collision energies as well as the optimization of collision energy. The method also detects low-abundance N-glycans more efficiently than MRM. This study is the first attempt to develop a sensitive PRM-based method to analyze permethylated N-glycans.

6-Plex mdSUGAR Isobaric-Labeling Guide Fingerprint Embedding for Glycomics Analysis

“…In addition, we also screened the fragmentation energy during PRM-MS/MS. As expected, lower normalized collision energy (NCE) prefers to produce larger B/Y ions for structure elucidation (Figure S8), which is consistent with a previous report, whereas higher NCE tends to fragment abundant signature oxonium ions such as m / z at 138, 168, 186, and 204 (for HexNAc) and 366 (for HexHexNAc) (Figure S9). Although these small fragments cannot piece together the whole precursor ion structure, they provide valuable information for the determination of whether a weak signal represents a glycan or not.…”

Development and application of GlycanDIA workflow for glycomic analysis

“…Although the parallel reaction monitoring (PRM) method was recently introduced to glycomic analysis, providing high sensitivity and high resolution, pre-knowledge about the analytes (such as target mass and retention time) is required, and only the ions within the inclusion list can be fragmented. 34 Meanwhile, GlycanDIA workflow owes the unique advantage that the dataset can continually be re-analyzed since all the precursors in the set mass ranges were fragmented during the data acquisition. This can provide valuable information by re-evaluating glycan modifications and discovering new glycan structures in future studies.…”

Fiber-Type Shifting in Sarcopenia of Old Age: Proteomic Profiling of the Contractile Apparatus of Skeletal Muscles

“…The verification phase of many proteomics investigations centers on confirming that the abundances of target peptides are significantly different between sample cohorts by using MS-derived quantitative measurements. Selected/Multiple-Reaction Monitoring (SRM/MRM) or Parallel-Reaction Monitoring (PRM) are examples of approaches that can be utilized, where precursor peptide ions are measured in predefined m/z and retention time [229][230][231]. Stable-isotope-labelled, synthetic peptides are often spiked into the samples of interest, a process that increases the overall accuracy of target peptide quantitation.…”