Simultaneous enrichment and fractionation of diverse proteins/peptides possessing different post-translational modifications (PTMs) from the same biological samples is highly desirable to reduce sample consumption, avoid complicated sample processing, and enable studies of potential crosstalks between different PTMs. In this work, we report a new approach to enable simultaneous enrichment and separation of glycopeptides, phosphopeptides, and mannose-6-phosphate (M6P) glycopeptides by using a dual-functional Ti(IV)-IMAC material. Moreover, we also made the separation of neutral and sialyl glycopeptides and mono- and multi-phosphopeptides possible by performing different elution processes according to the differences in their electrostatic or hydrophilic properties. These separations are effective and efficient to eliminate the signal suppression from neutral glycopeptides for sialyl glycopeptide detection, allowing separation of mono-phosphopeptides from multi-phosphopeptides, as well as detection of M6P glycopeptides that are free from the abovementioned modifications. This new strategy significantly improves the coverage and identification numbers of glycopeptides, phosphopeptides, and M6P glycopeptides by 1.9, 2.3, and 4.3-fold compared with the conventional method, respectively. This is the first report on simultaneous enrichment and separation of neutral and sialyl glycopeptides, mono- and multi-phosphopeptides, and M6P glycopeptides via dual-functional Ti(IV)- IMAC, revealing novel insights into potential crosstalk among these important PTMs.
Intact glycopeptide analysis has been of great interest because it can elucidate glycosylation site information and glycan structural composition at the same time. However, mass spectrometry (MS)-based glycoproteomic analysis is hindered by the low abundance and poor ionization efficiency of glycopeptides. Relatively large amounts of starting materials are needed for the enrichment, which makes the identification and quantification of intact glycopeptides from samples with limited quantity more challenging. To overcome these limitations, we developed an improved isobaric labeling strategy with an additional boosting channel to enhance N,N-dimethyl leucine (DiLeu) tagging-based quantitative glycoproteomic analysis, termed as Boost-DiLeu. With the integration of a one-tube sample processing workflow and high-pH fractionation, 3514 quantifiable N-glycopeptides were identified from 30 μg HeLa cell tryptic digests with reliable quantification performance. Furthermore, this strategy was applied to human cerebrospinal fluid (CSF) samples to differentiate N-glycosylation profiles between Alzheimer’s disease (AD) patients and non-AD donors. The results revealed processes and pathways affected by dysregulated N-glycosylation in AD, including platelet degranulation, cell adhesion, and extracellular matrix, which highlighted the involvement of N-glycosylation aberrations in AD pathogenesis. Moreover, weighted gene coexpression network analysis (WGCNA) showed nine modules of glycopeptides, two of which were associated with the AD phenotype. Our results demonstrated the feasibility of using this strategy for in-depth glycoproteomic analysis of size-limited clinical samples. Taken together, we developed and optimized a strategy for the enhanced comprehensive quantitative intact glycopeptide analysis with DiLeu labeling, showing significant promise for identifying novel therapeutic targets or biomarkers in biological systems with a limited sample quantity.
Site-specific O-glycoproteome mapping in complex biological systems provides a molecular basis for understanding the structure–function relationships of glycoproteins and their roles in physiological and pathological processes. Previous O-glycoproteome analysis in cerebrospinal fluid (CSF) focused on sialylated glycoforms, while missing information on other glycosylation types. In order to achieve an unbiased O-glycosylation profile, we developed an integrated strategy combining universal boronic acid enrichment, high-pH fractionation, and electron-transfer and higher-energy collision dissociation (EThcD) for enhanced intact O-glycopeptide analysis. We applied this strategy to analyze the O-glycoproteome in CSF, resulting in the identification of 308 O-glycopeptides from 110 O-glycoproteins, covering both sialylated and nonsialylated glycoforms. To our knowledge, this is the largest data set of O-glycoproteins and O-glycosites reported for CSF to date. We also developed a peptidomics workflow that utilized the EThcD and a three-step database searching strategy for comprehensive PTM analysis of endogenous peptides, including N-glycosylation, O-glycosylation, and other common peptide PTMs. Interestingly, among the 1411 endogenous peptides identified, 89 were O-glycosylated, and only one N-glycosylated peptide was found, indicating that CSF endogenous peptides were predominantly O-glycosylated. Analyses of the O-glycoproteome and endogenous peptidome PTMs were also conducted in the CSF of MCI and AD patients to provide a landscape of glycosylation patterns in different disease states. Our results showed a decreasing trend in fucosylation and an increasing trend of endogenous peptide O-glycosylation, which may play an important role in AD progression.
Graphical abstract The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) presents a serious threat to human health all over the world. The development of effective vaccines has been focusing on the spike (S) glycoprotein, which mediates viral invasion to human cells through its interaction with the angiotensin-converting enzyme 2 (ACE2) receptor. In this work, we perform analytical characterization of N- and O-linked glycosylation of the SARS-CoV-2 S glycoprotein. We explore the novel use of dual-functionalized titanium (IV)-immobilized metal affinity chromatography (Ti-IMAC) material for simultaneous enrichment and separation of neutral and sialyl glycopeptides of a recombinant SARS-CoV-2 S glycoprotein from HEK293 cells. This strategy helps eliminate signal suppression from neutral glycopeptides for the detection of sialyl glycopeptides and improves the glycoform coverage of the S protein. We profiled 19 of its 22 potential N-glycosylated sites with 398 unique glycoforms using the dual-functional Ti-IMAC approach, which exhibited improvement of coverage by 1.6-fold compared to the conventional hydrophilic interaction chromatography (HILIC) glycopeptide enrichment method. We also identified O-linked glycosylation site that was not found using the conventional HILIC approach. In addition, we reported on the identification of mannose-6-phosphate (M6P) glycosylation, which substantially expands the current knowledge of the spike protein’s glycosylation landscape and enables future investigation into the influence of M6P glycosylation of the spike protein on its cell entry. Supplementary Information The online version contains supplementary material available at 10.1007/s00216-021-03433-1.
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