title: Large-scale analysis of posttranslational modifications in the hippocampus of patients with Alzheimer's disease using pI shift and label-free quantification without enrichment.
abstract: Posttranslational modifications modulate protein function in cells. Global analysis of multiple posttranslational modifications can provide insight into physiology and disease, but presents formidable challenges. In the present study, we used a technique that does not require target enrichment to analyze alterations in the phosphorylation and ubiquitination of proteins from patients with Alzheimer's disease (AD). Guided by our previous findings, we applied three strategies to further our understanding of the dysregulation of posttranslationally modified proteins. We first identified phosphorylation sites by determining peptide pI shifts using OFFGEL. Second, using tandem mass spectrometry, we determined the ubiquitination status of the proteins using an assay for a trypsin digestion remnant of ubiquitination (Gly-Gly). Third, for large-scale discovery, we quantified the global differences in protein expression. Of the proteins expressed in AD tissue at levels of 2.0 or greater compared with controls, 60 were phosphorylated and 56 were ubiquitinated. Of the proteins expressed at levels of 0.5 or lower compared with controls, 81 were phosphorylated and 56 were ubiquitinated. Approximately 98 % of the phosphopeptides exhibited a pI shift. We identified 112 new phosphorylation sites (51.38 %), and 92 new ubiquitination sites (96.84 %). Taken together, our findings suggest that analysis of the alterations in posttranslationally modified proteins may contribute to understanding the pathogenesis of AD and other diseases.
authors.affiliation: Center for Cognition and Sociality, Institute for Basic Science, Daejeon, 305-811, Republic of Korea.
authors.forename: Taewook
authors.initials: T
authors.lastname: Kang
authors.forename: Jae Ho
authors.initials: JH
authors.lastname: Kim
authors.forename: Ingie
authors.initials: I
authors.lastname: Hong
authors.forename: Nam Hyun
authors.initials: NH
authors.lastname: Park
authors.forename: Nanhyun
authors.initials: N
authors.lastname: Park
authors.forename: Helmut
authors.initials: H
authors.lastname: Heinsen
authors.forename: Joo-Yong
authors.initials: JY
authors.lastname: Lee
authors.forename: Rivka
authors.initials: R
authors.lastname: Ravid
authors.forename: Isidro
authors.initials: I
authors.lastname: Ferrer
authors.forename: Jong Shin
authors.initials: JS
authors.lastname: Yoo
authors.forename: Kyung-Hoon
authors.initials: KH
authors.lastname: Kwon
authors.forename: Young Mok
authors.initials: YM
authors.lastname: Park
mesh_terms: D000367:Age Factors; D000368:Aged; D000369:Aged, 80 and over; D000444:Aldehyde Dehydrogenase / Q000737:chemistry; D000544:Alzheimer Disease / Q000503:physiopathology*; D001052:Apoferritins / Q000737:chemistry; D006624:Hippocampus / Q000378:metabolism* / Q000473:pathology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D050647:L-Aminoadipate-Semialdehyde Dehydrogenase; D008239:Lysine / Q000737:chemistry; D008875:Middle Aged; D009363:Neoplasm Proteins / Q000737:chemistry; D010455:Peptides / Q000737:chemistry; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D053719:Tandem Mass Spectrometry; D014357:Trypsin / Q000737:chemistry; D025801:Ubiquitin / Q000737:chemistry
chemical_list: D009363:Neoplasm Proteins; D010455:Peptides; D025801:Ubiquitin; D001052:Apoferritins; D000444:Aldehyde Dehydrogenase; C503677:AASDH protein, human; D050647:L-Aminoadipate-Semialdehyde Dehydrogenase; D014357:Trypsin; D008239:Lysine