MAGIC: An Automated N‑Linked Glycoprotein Identification Tool Using a Y1-Ion Pattern Matching Algorithm and in Silico MS2 Approach

• Published in: Analytical chemistry (Washington) Vol. 87; no. 4; pp. 2466 - 2473
• Main Authors: Lynn, Ke-Shiuan, Chen, Chen-Chun, Lih, T. Mamie, Cheng, Cheng-Wei, Su, Wan-Chih, Chang, Chun-Hao, Cheng, Chia-Ying, Hsu, Wen-Lian, Chen, Yu-Ju, Sung, Ting-Yi
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 17.02.2015
• Subjects: Algorithms; Automation; Computational Biology; data collection; Databases, Factual; Escherichia coli; Escherichia coli - chemistry; glycopeptides; Glycopeptides - analysis; Glycopeptides - chemistry; glycoproteins; glycosylation; HeLa Cells; Humans; ions; mass spectrometry; peroxidase; polysaccharides; proteome; Software
• ISSN: 0003-2700; 1520-6882; 1520-6882
• DOI: 10.1021/ac5044829

Glycosylation is a highly complex modification influencing the functions and activities of proteins. Interpretation of intact glycopeptide spectra is crucial but challenging. In this paper, we present a mass spectrometry-based automated glycopeptide identification platform (MAGIC) to identify peptide sequences and glycan compositions directly from intact N-linked glycopeptide collision-induced-dissociation spectra. The identification of the Y1 (peptideY0 + GlcNAc) ion is critical for the correct analysis of unknown glycoproteins, especially without prior knowledge of the proteins and glycans present in the sample. To ensure accurate Y1-ion assignment, we propose a novel algorithm called Trident that detects a triplet pattern corresponding to [Y0, Y1, Y2] or [Y0−NH3, Y0, Y1] from the fragmentation of the common trimannosyl core of N-linked glycopeptides. To facilitate the subsequent peptide sequence identification by common database search engines, MAGIC generates in silico spectra by overwriting the original precursor with the naked peptide m/z and removing all of the glycan-related ions. Finally, MAGIC computes the glycan compositions and ranks them. For the model glycoprotein horseradish peroxidase (HRP) and a 5-glycoprotein mixture, a 2- to 31-fold increase in the relative intensities of the peptide fragments was achieved, which led to the identification of 7 tryptic glycopeptides from HRP and 16 glycopeptides from the mixture via Mascot. In the HeLa cell proteome data set, MAGIC processed over a thousand MS2 spectra in 3 min on a PC and reported 36 glycopeptides from 26 glycoproteins. Finally, a remarkable false discovery rate of 0 was achieved on the N-glycosylation-free Escherichia coli data set. MAGIC is available at http://ms.iis.sinica.edu.tw/COmics/Software_MAGIC.html.