Glycosylation alterations in serum of Alzheimer's disease patients show widespread changes in N‐glycosylation of proteins related to immune function, inflammation, and lipoprotein metabolism

Abstract

Introduction There is an increased need for the development of novel blood‐based biomarkers for early detection, prevention, or intervention in Alzheimer's disease (AD). This study sought to determine whether serum glycopeptide analysis holds potential for identifying novel diagnostics and prognostics of AD. Methods The study involved 195 participants, including 96 patients with an AD diagnosis and 99 controls with no cognitive deficit. Utilizing a validated analytical mass spectrometry method, we monitored the site‐specific glycosylation of 52 serum glycoproteins. Results Partial least‐squares discriminant analysis revealed that changes in overall sialylation and fucosylation of serum glycoproteins may be indicators of an AD disease state. Loss of fucosylation of immunoglobulin G1 (IgG1) and IgG2 was indicative of AD diagnosis. Individual glycopeptide analysis found separation between the AD patients and controls on complement proteins and apolipoprotein B. Discussion The results of this study suggest that serum glycoprofiling may be a promising approach for biomarker discovery.

N-Glycan and Glycopeptide Serum Biomarkers in Philippine Lung Cancer Patients Identified Using Liquid Chromatography–Tandem Mass Spectrometry

“…Serum glycopeptides were prepared and analyzed as previously described, with modifications. − , Ten microliters (10 μL) each of serum samples, glycoprotein standardsimmunoglobulin M (IgM) and alpha-2-macroglobulin (A2MG)and standard serum (Sigma-Aldrich) were resolubilized in 50 μL buffer (50 mM NH 4 CO 3 ), reduced with 40 μL dithiothreitol (25 mM, 60 °C, 50 min), alkylated with 20 μL iodoacetamide (90 mM, 30 min), and then digested with 60 μL trypsin (0.067 μg/μL) overnight at 37 °C. After trypsin digestion, 20 μL of internal standard (peptide sequence: RPAIAINNPYVPR; 9 μg/mL) and 10 μL of 18% formic acid were added to quench the reaction.…”

“…To determine the linearity of the procedure, dilutions of digested standard serum samples were prepared (50–1500 μg/mL). Digested glycopeptides and peptides were analyzed in an Agilent 1290 infinity LC system coupled to an Agilent 6495 triple-quadrupole (QqQ) mass spectrometer (Santa Clara, CA) using a previously described dynamic multiple reaction monitoring method (dMRM). − , 2 μL of each sample was injected into an Agilent Eclipse plus C18 column (rapid resolution high definition [RRHD] 1.8 μm, 2.1 × 100 mm) coupled with a C18 guard column (RRHD 1.8 μm, 2.1 × 5 mm). Separation was performed using a binary solvent system composed of mobile phase A (3% v/v acetonitrile and 0.1% v/v formic acid in water) and mobile phase B (90% v/v acetonitrile and 0.1% v/v formic acid in water).…”

“…The gradient sequence for separation used was as follows: 0–2.5 min, 1% B; 2.5–20 min, 16% B; 20–35 min, 58% B; 35–40 min, 100% B; 40–50 min, 100% B; and 50.01–65 min, 0% B with a flow rate of 0.5 mL/min. The dMRM method applied predetermined CID energies to detect and quantify 505 specific glycopeptide transitions . The dMRM results were analyzed using MassHunter Quantitative Analysis Software B.08.00 (Agilent Technologies).…”

“…The dMRM method applied predetermined CID energies to detect and quantify 505 specific glycopeptide transitions. 28 The dMRM results were analyzed using MassHunter Quantitative Analysis Software B.08.00 (Agilent Technologies). Data was exported as integrated areas, and glycopeptide abundance was normalized to corresponding peptide concentrations to reduce the variability caused by over- or underexpression of proteins.…”

Identification and immune characteristics of molecular subtypes related to protein glycosylation in Alzheimer’s disease

“…Additionally, protein glycosylation is closely related to immune cells and immune response in AD. Some studies have found that most of the glycoproteins with site-specific glycosylation in the serum of patients with AD are involved in immune function and induce specific inflammatory pathways and immune responses ( Tena et al, 2022 ). Although existing studies have preliminarily revealed the relationship of protein glycosylation and protein glycosylation-related genes (PGRGs) with AD, there is still a lack of comprehensive analysis of the role of PGRGs in the occurrence and development of AD combined with immune imbalance.…”