Classification of Tandem Mass Spectra for Identification of N- and O-linked Glycopeptides.

Shadi Toghi Eshghi,Yingwei Hu,Xingde Li,Punit Shah,Weiming Yang,Shisheng Sun,Hui Zhang

doi:10.1038/srep37189

Shadi Toghi Eshghi, Yingwei Hu + Show 5 more

Open Access

https://doi.org/10.1038/srep37189

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Journal: Scientific Reports	Publication Date: Nov 21, 2016
Citations: 47	License type: CC BY 4.0

Affiliation: Johns Hopkins University

Abstract

Analysis of intact glycopeptides by mass spectrometry is essential to determining the microheterogeneity of protein glycosylation. Higher-energy collisional dissociation (HCD) fragmentation of glycopeptides generates mono- or disaccharide ions called oxonium ions that carry information about the structure of the fragmented glycans. Here, we investigated the link between glycan structures and the intensity of oxonium ions in the spectra of glycopeptides and utilized this information to improve the identification of glycopeptides in biological samples. Tandem spectra of glycopeptides from fetuin, glycophorin A, ovalbumin and gp120 tryptic digests were used to build a spectral database of N- and O-linked glycopeptides. Logistic regression was applied to this database to develop model to distinguish between the spectra of N- and O-linked glycopeptides. Remarkably, the developed model was found to reliably distinguish between the N- and O-linked glycopeptides using the spectral features of the oxonium ions using verification spectral set. Finally, the performance of the developed predictive model was evaluated in HILIC enriched glycopeptides extracted from human serum. The results showed that pre-classification of tandem spectra based on their glycosylation type improved the identification of N-linked glycopeptides. The developed model facilitates interpretation of tandem mass spectrometry data for assignment of glycopeptides.

Highlights

The ability of cells to co- and post-translationally modify proteins has created tremendous diversity in the protein forms and functions
Excluding the oxonium ion intensities ensures that these ions, which are disproportionately abundant in the spectra of N-glycopeptides, do not interfere with the scoring of the glycopeptide-spectral matches (GPSM)
An important signature of glycopeptide spectra is the presence of oxonium ions that are mono- or disaccharides fragmented from the glycan moieties

Summary

Introduction

The ability of cells to co- and post-translationally modify proteins has created tremendous diversity in the protein forms and functions. Excluding the oxonium ion intensities ensures that these ions, which are disproportionately abundant in the spectra of N-glycopeptides, do not interfere with the scoring of the GPSMs. Data analysis of human serum samples. Concatenation of the data from ovalbumin, glycophorin A, fetuin and gp[120] resulted in a training dataset containing 872 and 527 HCD fragmented spectra of N- and O-glycopeptides, respectively (supplementary Table S1).

Results

Conclusion