In-depth Site-specific Analysis of N-glycoproteome in Human Cerebrospinal Fluid and Glycosylation Landscape Changes in Alzheimer's Disease.

As the body fluid that directly interchanges with the extracellular fluid of the central nervous system (CNS), cerebrospinal fluid (CSF) serves as a rich source for CNS-related disease biomarker discovery. Extensive proteome profiling has been conducted for CSF, but studies aimed at unraveling site-specific CSF N-glycoproteome are lacking. Initial efforts into site-specific N-glycoproteomics study in CSF yield limited coverage, hindering further experimental design of glycosylation-based disease biomarker discovery in CSF. In the present study, we have developed an N-glycoproteomic approach that combines enhanced N-glycopeptide sequential enrichment by hydrophilic interaction chromatography (HILIC) and boronic acid enrichment with electron transfer and higher-energy collision dissociation (EThcD) for large-scale intact N-glycopeptide analysis. The application of the developed approach to the analyses of human CSF samples enabled identifications of a total of 2893 intact N-glycopeptides from 511 N-glycosites and 285 N-glycoproteins. To our knowledge, this is the largest site-specific N-glycoproteome dataset reported for CSF to date. Such dataset provides molecular basis for a better understanding of the structure-function relationships of glycoproteins and their roles in CNS-related physiological and pathological processes. As accumulating evidence suggests that defects in glycosylation are involved in Alzheimer's disease (AD) pathogenesis, in the present study, a comparative in-depth N-glycoproteomic analysis was conducted for CSF samples from healthy control and AD patients, which yielded a comparable N-glycoproteome coverage but a distinct expression pattern for different categories of glycoforms, such as decreased fucosylation in AD CSF samples. Altered glycosylation patterns were detected for a number of N-glycoproteins including alpha-1-antichymotrypsin, ephrin-A3 and carnosinase CN1 etc., which serve as potentially interesting targets for further glycosylation-based AD study and may eventually lead to molecular elucidation of the role of glycosylation in AD progression.

Simultaneous enrichment and separation of neutral and sialyl glycopeptides of SARS-CoV-2 spike protein enabled by dual-functionalized Ti-IMAC material.

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.

In-Depth Site-Specific O-Glycosylation Analysis of Glycoproteins and Endogenous Peptides in Cerebrospinal Fluid (CSF) from Healthy Individuals, Mild Cognitive Impairment (MCI), and Alzheimer's Disease (AD) Patients.

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.

Activity of Phosvitin in Hydroxyapatite Acid-Damage Immersion and Antimicrobial Assays.

Phosvitin, the most highly phosphorylated metal-binding protein found in nature, binds more than 100 calcium ions, and has been identified as an agent that could be used to generate biomineralization scaffolds. Because of published reports describing phosvitin's affinity for calcium and potential antibiotic activity, this study was undertaken in order to evaluate phosvitin for both antibiotic activity against common microorganisms and the ability to protect hydroxyapatite surfaces from acid damage. To more clearly define its antibiotic action, the effects of phosvitin on Micrococcus luteus, P. mirabilis, B. cereus, E. coli, and S. epidermidis were evaluated. In both Kirby-Bauer tests and liquid culture growth inhibition assays, phosvitin inhibited M. luteus, a microorganism that thrives in the human mouth, but not the other bacteria tested. The MIC of phosvitin was determined to be 31.3 µg/mL when delivered in 1 mM CaCl2 but was 0.5 mg/mL in the absence of added calcium. Expanding on the potential impacts of phosvitin on the mouth, its action was evaluated in a model of tooth decay represented by acid-damaged hydroxyapatite discs. SEM, AFM, and FAAS analyses revealed that pretreatment of discs with phosvitin modulated the damage-induced morphology and topography changes associated with acid-damaged discs.