Multilayered N-Glycoproteome Profiling Reveals Highly Heterogeneous and Dysregulated Protein N-Glycosylation Related to Alzheimer’s Disease

Abstract

Protein N-glycosylation is ubiquitous in the brain and is closely related to cognition and memory. Alzheimer’s disease (AD) is a multifactorial disorder that lacks a clear pathogenesis and treatment. Aberrant N-glycosylation has been suggested to be involved in AD pathology. However, the systematic variations in protein N-glycosylation and their roles in AD have not been thoroughly investigated due to technical challenges. Here, we applied multilayered N-glycoproteomics to quantify the global protein expression levels, N-glycosylation sites, N-glycans, and site-specific N-glycopeptides in AD (APP/PS1 transgenic) and wild-type mouse brains. The N-glycoproteomic landscape exhibited highly complex site-specific heterogeneity in AD mouse brains. The generally dysregulated N-glycosylation in AD, which involved proteins such as glutamate receptors as well as fucosylated and oligomannose glycans, were explored by quantitative analyses. Furthermore, functional studies revealed the crucial effects of N-glycosylation on proteins and neurons. Our work provides a systematic multilayered N-glycoproteomic strategy for AD and can be applied to diverse biological systems.

A streamlined pipeline for multiplexed quantitative site-specific N-glycoproteomics

“…For proteomics analysis on Orbitrap Fusion, the following settings were used. MS1 settings: Detector Type-Orbitrap, Orbitrap Resolution-120 k, Scan Range-350–1,550, Maximum injection time-50 ms, AGC target-4e 5 , RF Lens-60%, and Data Type-Profile. MS2 settings: Isolation mode-Quadrupole, Isolation window-1.6 m/z , Scan range mode-Auto normal, First mass-110, Activation type-HCD, Collision energy (%)−40, Detector type-Orbitrap, Orbitrap resolution-60 K, Maximum injection time-60 ms, AGC target-5e 4 , and Data type-Profile.…”

“…Conventional analytical approaches often release the glycan moiety chemically or enzymatically from proteins before performing MS analysis of either the resulting oligosaccharides or the de-glycosylated peptides 3 , thus deliberately discarding information about site specificity. Instead, analysis of intact N -glycopeptides (i.e., peptides that derive from endoproteolytic digestion of glycosylated proteins, but still carry the intact glycan moieties) is essential to determine the linkage between protein and glycan, and to profile the microheterogeneity of glycosylation at each site 4 , 5 . Significant improvements have been made with regard to methods for intact glycopeptide characterization, including glycopeptide enrichment 6 – 8 , optimized MS acquisition strategies 8 , advanced fragmentation techniques 9 , 10 , and database search algorithms 11 .…”

SugarQuant: a streamlined pipeline for multiplexed quantitative site-specific N-glycoproteomics

“…For proteomics analysis on Orbitrap Fusion, the following settings were used. MS1 settings: Detector Type-Orbitrap, Orbitrap Resolution-120 k, Scan Range-350-1,550, Maximum injection time-50 ms, AGC target-4e 5 For the evaluation of IgM N-glycopeptide HCD fragmentation, each selected precursor in MS1 survey scans was subject to 8 consecutive MS2 scans with NCE15%, 20%, 25%, 30%, 35%, 40%, 45%, and 50%, respectively ( Fig. 2a, Supp.…”

“…Conventional analytical approaches often release the glycan moiety chemically or enzymatically from proteins before performing MS analysis of either the resulting oligosaccharides or the de-glycosylated peptides 3 , thus deliberately discarding information about site specificity. Instead, analysis of intact N-glycopeptides (i.e., peptides that derive from endoproteolytic digestion of glycosylated proteins, but still carry the intact glycan moieties) is essential to determine the linkage between protein and glycan, and to profile the micro-heterogeneity of glycosylation at each site 4,5 . Significant improvements have been made with regard to methods for intact glycopeptide characterization, including glycopeptide enrichment [6][7][8] , optimized MS acquisition strategies 8 , advanced fragmentation techniques 9, 10 and database search algorithms 11 .…”

Optimal Dissociation Methods Differ for N- and O-glycopeptides

“…In 2017, Liu et al used sceHCD methods for identification of ~10,000 N-glycosites from five mouse tissues, 78 which contributed to its popularity in recent N-glycopeptide analysis. [79][80][81][82][83][84][85][86] A few studies have looked to extend the application of sceHCD to O-glycopeptides, 75,76,79,87,88 but this has not been as widespread. Limited comparisons between sceHCD and ETD methods have been performed for N-glycopeptides, 78 as have comparisons of HCD and EThcD for Oglycopeptides; 64,69 however, a comprehensive head-to-head comparison of standard HCD, sceHCD, ETD, and EThcD has not been reported.…”