Proteome analysis of human metaphase chromosomes

Susumu Uchiyama, Shouhei Kobayashi, Hideaki Takata, Takeshi Ishihara, Naoto Hori, Tsunehito Higashi, Kayoko Hayashihara, Takefumi Sone, Daisuke Higo, Takashi Nirasawa, Toshifumi Takao, Sachihiro Matsunaga, Kiichi Fukui. Proteome Analysis of Human Metaphase Chromosomes. Journal of Biological Chemistry, Volume 280, Issue 17, 2005, Pages 16994-17004. https://doi.org/10.1074/jbc.M412774200

Hornerin deposits in neuronal intranuclear inclusion disease : direct identification of proteins with compositionally biased regions in inclusions

Neuronal intranuclear inclusion disease (NIID) is a neurodegenerative disorder, characterized by the presence of eosinophilic inclusions (NIIs) within nuclei of central and peripheral nervous system cells. This study aims to identify the components of NIIs, which have been difficult to analyze directly due to their insolubility. In order to establish a method to directly identify the components of NIIs, we first analyzed the huntingtin inclusion-rich fraction obtained from the brains of Huntington disease model mice. Although the sequence with expanded polyglutamine could not be identified by liquid-chromatography mass spectrometry, amino acid analysis revealed that glutamine of the huntingtin inclusion-rich fraction increased significantly. This is compatible with the calculated amino acid content of the transgene product. Therefore, we applied this method to analyze the NIIs of diseased human brains, which may have proteins with compositionally biased regions, and identified a serine-rich protein called hornerin. Since the analyzed NII-rich fraction was also serine-rich, we suggested hornerin as a major component of the NIIs. A specific distribution of hornerin in NIID was also investigated by Matrix-assisted laser desorption/ionization imaging mass spectrometry and immunofluorescence. Finally, we confirmed a variant of hornerin by whole-exome sequencing and DNA sequencing. This study suggests that hornerin may be related to the pathological process of this NIID, and the direct analysis of NIIs, especially by amino acid analysis using the NII-rich fractions, would contribute to a deeper understanding of the disease pathogenesis.Peer reviewe

Charged chiral derivatization for enantioselective imaging of d-,l-2-hydroxyglutaric acid using ion mobility spectrometry/mass spectrometry

A newly synthesized charged chiral tag-enabled enantioselective imaging of D-,L-2-hydroxyglutaric acid, which are independently associated with the regulation of DNA methylation. The tag-conjugated diastereomers were ionized efficiently through MALDI, separated by ion mobility spectrometry, and further separated from other molecules in mass spectrometry. On-tissue chiral derivatization using the tag facilitated the visualization of different distributions of the two isomers in the mouse testis.journal articl

Charged Chiral Derivatization for Enantioselective Imaging of D-, L-2-Hydroxyglutaric Acid Using Ion Mobility Spectrometry/Mass Spectrometry

A newly synthesized charged chiral tag enabled enantioselective imaging of D-, L-2-hydroxyglutaric acid, which are independently associated with the regulation of DNA methylation. The tag-conjugated diastereomers were ionized efficiently through MALDI, separated by ion mobility spectrometry, and further separated from other molecules in mass spectrometry. On-tissue chiral derivatization using the tag facilitated the visualization of different distributions of the two isomers in the mouse testis

Distinct deposition of amyloid-β species in brains with Alzheimer’s disease pathology visualized with MALDI imaging mass spectrometry

Abstract Amyloid β (Aβ) deposition in the brain is an early and invariable feature of Alzheimer’s disease (AD). The Aβ peptides are composed of about 40 amino acids and are generated from amyloid precursor proteins (APP), by β- and γ-secretases. The distribution of individual Aβ peptides in the brains of aged people, and those suffering from AD and cerebral amyloid angiopathy (CAA), is not fully characterized. We employed the matrix-assisted laser desorption/ionization-imaging mass spectrometry (MALDI-IMS) to illustrate the spatial distribution of a broad range of Aβ species in human autopsied brains. With technical advancements such as formic acid pretreatment of frozen autopsied brain samples, we have: i) demonstrated that Aβ1–42 and Aβ1–43 were selectively deposited in senile plaques while full-length Aβ peptides such as Aβ1–36, 1–37, 1–38, 1–39, 1–40, and Aβ1–41 were deposited in leptomeningeal blood vessels. ii) Visualized distinct depositions of N-terminal truncated Aβ40 and Aβ42, including pyroglutamate modified at Glu-3 (N3pE), only with IMS for the first time. iii) Demonstrated that one single amino acid alteration at the C-terminus between Aβ1–42 and Aβ1–41 results in profound changes in their distribution pattern. In vitro, this can be attributed to the difference in the self-aggregation ability amongst Aβ1–40, Aβ1–41, and Aβ1–42. These observations were further confirmed with immunohistochemistry (IHC), using the newly developed anti-Aβ1–41 antibody. Here, distinct depositions of truncated and/or modified C- and N-terminal fragments of Aβs in AD and CAA brains with MALDI-IMS were visualized in a spacio-temporal specific manner. Specifically, Aβ1–41 was detected both with MALDI-IMS and IHC suggesting that a single amino acid alteration at the C-terminus of Aβ results in drastic distribution changes. These results suggest that MALDI-IMS could be used as a standard approach in combination with clinical, genetic, and pathological observations in understanding the pathology of AD and CAA