Open AccessData-Independent Acquisition-Based Mass Spectrometry (DIA-MS) for Quantitative Analysis of Intact N-Linked Glycopeptides
Author(s) -
Mingming Dong,
Tung Shing Mamie Lih,
Minghui Ao,
Yingwei Hu,
Shao Yung Chen,
Rodrigo Vargas Eguez,
Hui Zhang
Publication year - 2021
Publication title -
analytical chemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.117
H-Index - 332
eISSN - 1520-6882
pISSN - 0003-2700
DOI - 10.1021/acs.analchem.1c01659
Subject(s) - glycopeptide , chemistry , glycosylation , mass spectrometry , chromatography , proteomics , computational biology , biochemistry , biology , gene , antibiotics
N-linked protein glycosylation is a key regulator in various biological functions. Previous studies have shown that aberrant glycosylation is associated with many diseases. Therefore, it is essential to elucidate protein modifications of glycosylation by quantitatively profiling intact N-linked glycopeptides. Data-independent acquisition (DIA) mass spectrometry (MS) is a cost-effective, flexible, and high-throughput method for global proteomics. However, substantial challenges are still present in the quantitative analysis of intact glycopeptides with high accuracy at high throughput. In this study, we have established a novel integrated platform for the DIA analysis of intact glycopeptides isolated from complex samples. The established analysis platform utilizes a well-designed DIA-MS method for raw data collection, a spectral library constructed specifically for intact glycopeptide quantification providing accurate results by the inclusion of Y ions for quantification and filtering of quantified intact glycopeptides with low-quality MS2 spectra automatically using a set of criteria. Intact glycopeptides isolated from human serum were used to evaluate the performance of the integrated platform. By utilizing 100 isolation windows for DIA data acquisition, a well-constructed human serum spectral library containing 1123 nonredundant intact glycopeptides with Y ions, and automated data inspection, 620 intact glycopeptides were quantified with high confidence from DIA-MS. In summary, our integrated platform can serve as a reliable quantitative tool for characterizing intact glycopeptides isolated from complex biological samples to assist our understanding of biological functions of N-linked glycosylation.