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.