Analyzing large-scale intact glycopeptides presents significant challenges due to the complex nature of glycopeptide structures and the intricacies of data analysis. The thoroughness of glycopeptide identification is heavily dependent on the glycan library used during data searches, which can result in some glycopeptides being overlooked. In this study, we examine our ability to detect novel glycopeptides that were previously missed in published datasets. We provide notable examples of identifying phosphorylated glycopeptides and acetylated sialoglycopeptides that were not detected in initial searches.
Moreover, we compare the performance of the Orbitrap Eclipse and Orbitrap Ascend Tribrid mass spectrometers for glycopeptide analysis. Our results demonstrate that the Orbitrap Ascend Tribrid MS nearly doubles the identification rate compared to the Orbitrap Eclipse Tribrid MS, with a remarkable 99% overlap of glycopeptides identified by the Orbitrap Eclipse. We also present large-scale glycoproteomics studies carried out using the new Orbitrap Astral mass spectrometer. A glycoproteomics study utilizing HCD stepped collision energy on the Orbitrap Astral showed a speed increase of up to 3x compared to the Orbitrap Exploris 480 MS. Using two or three collision energies, rather than just one, significantly improved the confidence in glycopeptide identifications.
All the data in these experiments were enriched and analyzed using the Orbitrap Eclipse, Orbitrap Ascend Tribrid, and Orbitrap Astral mass spectrometers. The data analyses were meticulously performed using Byonic software (Protein Metrics), with some of the raw files sourced from publicly available databases (MSV000091467, MSV000094280).