Systematic Uncovering of Multiple Pathways Underlying the Pathology of Huntington Disease by an Acid-cleavable Isotope-coded Affinity Tag Approach

Abstract

Huntington disease (HD) is an autosomal dominant neurodegenerative disease that results from a CAG (glutamine) trinucleotide expansion in exon 1 of huntingtin (Htt). The aggregation of mutant Htt has been implicated in the progression of HD. The earliest degeneration occurs in the striatum. To identify proteins critical for the progression of HD, we applied acid-cleavable ICAT technology to quantitatively determine changes in protein expressions in the striatum of a transgenic HD mouse model (R6/2). The cysteine residues of striatal proteins from HD and wild-type mice were labeled, respectively, with the heavy and light forms of the ICAT reagents. Samples were trypsinized, uncovered by avidin affinity chromatography, and analyzed by nano-LC-MS/MS. Western blot analyses were used to confirm and to calibrate the ICAT ratios. Linear regression was used to uncover a group of proteins that exhibited consistent changes. In two independent ICAT experiments, we identified 427 cysteine-containing striatal proteins among which ϳ66% (

Quantitative Proteomics Analysis of the Effects of Ionizing Radiation in Wild Type and p53K317R Knock-in Mouse Thymocytes

“…Of the proteins identified and quantified, 69% were observed in more than one replicate and 68% were identified by two or more unique peptides. The latter proportion is consistent with other studies using quantification based on residuespecific labeling (25). Of the proteins quantified, the relative amounts of 102 proteins (9.2%) were significantly changed by exposure to IR (p Ͻ 0.05).…”

Quantitative Proteomics of a Presymptomatic A53T α-Synuclein Drosophila Model of Parkinson Disease

“…For example, the ribosomal protein S3A-derived peptide ion [LLELHGDGGGK ϩ 2H] 2ϩ identified only in the forward labeling experiment showed a 0.59-fold change, whereas the peptide ion [IYPLHDVYIR ϩ 2H] 2ϩ identified only in the reverse labeling experiment exhibited a 1.98-fold change. Similar discrepancies in peptide foldchange values have also been observed using an isotopecoded affinity tag approach (40). As discussed previously by others, poor agreement in relative abundance of different peptides from the same protein may also arise due to posttranslational modifications (39).…”

“…Currently, various criteria have been utilized to determine differential expression of proteins based on data quality (39 -43). For instance, Griffin et al (43) applied a 1.5-fold difference criterion from the ICAT method as a threshold indicating significant change, and Chiang et al (40) considered a protein with a calibrated ratio (ICAT analyses calibrated by Western blot analysis) of either Ն1.23 or Յ0.77 to be differentially expressed. The present study utilized three proteins as internal standards at different concentration ratios at an early stage of sample preparation to estimate experimental variability, e.g.…”

The Role of Histone Deacetylases in Neurodegenerative Diseases and Small-Molecule Inhibitors as a Potential Therapeutic Approach

“…The same holds true for the polyQ diseases: in HD, transcriptional profiling of human tissue derived from HD-affected individuals shows an aberrant expression of a large number of genes and proteins [145][146][147]. Characteristic gene expression changes have also been observed in various mouse models designed to replicate certain aspects of the disease [147][148][149][150][151]. Similarly, mice overexpressing polyQ-expanded atrophin (a model of DRPLA) [152], expanded ataxin-7 [152,153], and the mutant androgen receptor [152] also show aberrant gene expression.…”