Compared to previously reported atypical N glycosites that w

Compared to previously reported atypical N-glycosites that were identified based on the deamidation of asparagine residues after PNGase F treatment [106], [107], these two studies further validated the identified atypical motif glycosites by directly identifying their intact glycopeptides. Since the deamidation of N can sometimes cause false-positives, direct identification of the intact glycopeptides with glycan attached is particularly important for confirmation of these atypical N-glycosylation sites detected by mass spectrometry-based glycoproteomics.
Future perspectives Owing to the huge improvement of mass spectrometry technology, liquid chromatography separation, analysis software and glycoprotein/glycosite-containing peptide isolation methods in recent years, many mass spectrometry methods and strategies have been developed for large-scale profiling of N-linked neuropeptides and glycosylation sites. Prior to MS analysis, glycopeptide enrichment is usually performed to increase the concentration of the glycopeptides. Then glycans are normally removed from the glycosite-containing peptides to eliminate the micro-heterogeneity of the glycosylation. By using this strategy, large-scale glycosylation analysis has been achieved by employing proteomic approaches, and the glycan attached sites can be identified through the deamidation (with +0.98 Da shift) at the formerly glycosylation sites [7], [8]. The majority of human N-linked glycoproteins and glycosylation sites known nowadays were identified though this strategy. This strategy, however, would cause false identification when the mass accuracy of the detected peptide is not high enough. The high resolution and accuracy of mass spectrometers developed in recent years have greatly increased the identification confidence of the glycosites and glycoproteins, and increased the numbers of identified glycosite-containing peptides, but the false positive rates of identified glycosylation sites using this strategies are still much higher than we expected, as deamidation of asparagine could be induced during sample preparation, and even occur naturally in human body, as reported in recent studies [108], [109]. The naturally occurred deamidation can be influenced by several factors, such as pH (in particular pH conditions for PNGase F driven deglycosylation), temperature, ionic strength, and others [108]. Therefore, appropriate controls and extreme care have to be taken in both sample preparation and data interpretation to prevent false-positive identifications [109]. Based on the data summarized above, although several thousand glycoproteins could be identified from one single body fluid or tissue, there are still many tissues (such as heart, stomach, brain, and kidney) in which no mass spectrometry-based glycosite data has been generated yet. In addition, the identification numbers of glycoproteins and glycosylation sites are largely determined by the approaches of sample preparation, mass spectrometry analysis as well as data analysis. Therefore, a systematically glycoproteomic analysis of various human body fluids and tissues using a uniform platform may still be needed. This uniform platform may need to combine several different strategies, such as using different enzymatic digestion [34], enrichment methods, as well as intact glycopeptide analysis, to maximize the identification coverage of human glycoproteins and glycosites. Such work will help to create a high quality of human glycoproteome database to facilitate the future studies of glycoprotein structures and functions. Along with glycosylation sites, characterization of the glycans attached is also essential for the understanding the biological roles of a glycoprotein. Although the overall glycoform diversities can be revealed by analyzing the released glycans or de-glycosylated peptides, the “true” glycan heterogeneity still need to be uncovered at the intact glycopeptides or glycoproteins level. In recent years, direct analysis of intact glycopeptide/glycoproteins was enabled with the development of mass spectrometry technologies and softwares [84], [112], [113], [114], [115], [116]. Detailed information is available from reviews by Cao et al., 2016 and Yang et al., 2017 [117], [118]. Intact glycopeptide analysis can provide additional site-specific glycosylation information, which allows us to know which glycans are attached to which glycosylation sites [84]. These technologies will indisputably push glycoproteomic researches to a higher stage.