Human Alzheimer's disease synaptic O-GlcNAc site mapping and iTRAQ expression proteomics with ion trap mass spectrometry.
Neuronal synaptic functional deficits are linked to impaired learning and memory in Alzheimer's disease (AD). We recently demonstrated that O-GlcNAc, a novel cytosolic and nuclear carbohydrate post-translational modification, is enriched at neuronal synapses and positively regulates synaptic plasticity linked to learning and memory in mice. Reduced levels of O-GlcNAc have been observed in AD, suggesting a possible link to deficits in synaptic plasticity. Using lectin enrichment and mass spectrometry, we mapped several human cortical synaptic O-GlcNAc modification sites. Overlap in patterns of O-GlcNAcation between mouse and human appears to be high, as previously mapped mouse synaptic O-GlcNAc sites in Bassoon, Piccolo, and tubulin polymerization promoting protein p25 were identified in human. Novel O-GlcNAc modification sites were identified on Mek2 and RPN13/ADRM1. Mek2 is a signaling component of the Erk 1/2 pathway involved in synaptic plasticity. RPN13 is a component of the proteasomal degradation pathway. The potential interplay of phosphorylation with mapped O-GlcNAc sites, and possible implication of those sites in synaptic plasticity in normal versus AD states is discussed. iTRAQ is a powerful differential isotopic quantitative approach in proteomics. Pulsed Q dissociation (PQD) is a recently introduced fragmentation strategy that enables detection of low mass iTRAQ reporter ions in ion trap mass spectrometry. We optimized LTQ ion trap settings for PQD-based iTRAQ quantitation and demonstrated its utility in O-GlcNAc site mapping. Using iTRAQ, abnormal synaptic expression levels of several proteins previously implicated in AD pathology were observed in addition to novel changes in synaptic specific protein expression including Synapsin II.
Skorobogatko
Yuliya V
YV
Department of Biochemistry and Molecular Biology, Drexel University College of Medicine, Philadelphia, PA, USA.
Deuso
John
J
Adolf-Bryfogle
Jared
J
Adolf-Bergfoyle
Jared
J
Nowak
Matthew G
MG
Gong
Yuesong
Y
Lippa
Carol Frances
CF
Vosseller
Keith
K
D000117:Acetylglucosamine / Q000378:metabolism*; D000544:Alzheimer Disease / Q000235:genetics* / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D002540:Cerebral Cortex / Q000737:chemistry / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D013058:Mass Spectrometry; D051379:Mice; D008969:Molecular Sequence Data; D009473:Neuronal Plasticity; D010449:Peptide Mapping; D010766:Phosphorylation; D011506:Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D016415:Sequence Alignment; D013569:Synapses / Q000737:chemistry* / Q000378:metabolism*
D011506:Proteins; D000117:Acetylglucosamine