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"Ghost" Fragment Ions in Structure and Site-Specific Glycoproteomics Analysis
Diana Campos1, Michael Girgis2, Qiang Yang3
1Max-Planck-Institut fuer Herz- und Lungenforschung, Ludwigstrasse 43, 61231 Bad Nauheim, Germany.
Analytical Chemistry
|June 29, 2023
Summary
Glycoproteomics analysis using mass spectrometry faces challenges with complex glycan structures. This study identifies "Ghost" fragments that cause false positives and proposes a threshold to improve accuracy in N-glycopeptide analysis.
Area of Science:
- Glycomics
- Proteomics
- Mass Spectrometry
Background:
- Glycosylation analysis via mass spectrometry (MS) is vital for understanding glycoproteins.
- Distinguishing isobaric glycopeptide structures is a major challenge in glycoproteomics.
- Collision energy (CE) modulation has shown promise for qualitative glycan structure elucidation.
Purpose of the Study:
- To investigate the specificity of glycopeptide fragmentation patterns in N-glycoproteomics.
- To identify and characterize potential sources of false-positive structure assignments.
- To develop methods for improving the accuracy of glycopeptide analysis.
Main Methods:
- Utilized synthetic stable isotope-labeled N-glycopeptide standards for N-glycoproteomics analysis.
- Investigated fragmentation specificity using collision-induced dissociation (CID) and higher-energy collisional dissociation (HCD) fragmentation.
- Analyzed fragments originating from both the oligomannose core and outer antennary structures.
Main Results:
- Identified "Ghost" fragments resulting from glyco unit rearrangement or mannose core fragmentation, leading to potential false-positive structure assignments.
- Demonstrated that different glycan linkages exhibit varying stabilities under fragmentation conditions.
- Established a minimal intensity threshold for specific fragments to prevent misidentification.
Conclusions:
- The study provides a method to mitigate false-positive assignments in glycoproteomics.
- Implementing intensity thresholds for specific fragments enhances the reliability of N-glycopeptide structure determination.
- Findings contribute to more accurate and dependable glycoproteomics measurements.
