Characterization and identification of dityrosine cross-linked peptides using tandem mass spectrometry

The use of mass spectrometry coupled with chemical cross-linking of proteins has become a powerful tool for proteins structure and interactions studies. Unlike structural analysis of proteins using chemical reagents specific for lysine or cysteine residues, identification of gas-phase fragmentation patterns of endogenous dityrosine cross-linked peptides have not been investigated. Dityrosine cross-linking in proteins and peptides are clinical markers of oxidative stress, aging, and neurodegenerative diseases including Alzheimer’s disease and Parkinson’s disease. In this study, we investigated and characterized the fragmentation pattern of a synthetically prepared dityrosine cross-linked dimer of Aβ(1–16) using ESI tandem mass spectrometry. We then detailed the fragmentation pattern of dityrosine cross-linked Aβ(1–16), using collision induced dissociation (CID), higher-energy collision induced dissociation (HCD), electron transfer dissociation (ETD), and electron capture dissociation (ECD). Application of these generic fragmentation rules of dityrosine cross-linked peptides allowed for the identification of dityrosine cross-links in peptides of Aβ and α-synuclein generated in vitro by enzymatic peroxidation. We report, for the first time, the dityrosine cross-linked residues in human hemoglobin and α-synuclein under oxidative conditions. Together these tools open up the potential for automated analysis of this naturally occurring post-translation modification in neurodegenerative diseases as well as other pathological conditions

Production of human skeletal α-actin proteins by the baculovirus expression system

Mutations within the human skeletal muscle α-actin gene cause three different skeletal muscle diseases. Functional studies of the mutant proteins are necessary to better understand the pathogenesis of these diseases, however, no satisfactory system for the expression of mutant muscle actin proteins has been available. We investigated the baculovirus expression vector system (BEVS) for the abundant production of both normal and mutant skeletal muscle α-actin. We show that non-mutated actin produced in the BEVS behaves similarly to native actin, as shown by DNase I affinity purification, Western blotting, and consecutive cycles of polymerisation and depolymerisation. Additionally, we demonstrate the production of mutant actin proteins in the BEVS, without detriment to the insect cells in which they are expressed. The BEVS therefore is the method of choice for studying mutant actin proteins causing human diseases

A novel association between a SNP in CYBRD1 and serum ferritin levels in a cohort study of HFE hereditary haemochromatosis

There is emerging evidence that there are genetic modifiers of iron indices for HFE gene mutation carriers at risk of hereditary hemochromatosis. A random sample, stratified by HFE genotype, of 863 from a cohort of 31 192 people of northern European descent provided blood samples for genotyping of 476 single nucleotide polymorphisms (SNPs) in 44 genes involved in iron metabolism. Single SNP association testing, using linear regression models adjusted for sex, menopause and HFE genotype, was conducted for four continuously distributed outcomes: serum ferritin (log transformed), transferrin saturation, serum transferrin, and serum iron. The SNP rs884409 in CYBRD1 is a novel modifier specific to HFE C282Y homozygotes. Median unadjusted serum ferritin concentration decreased from 1194 microg/l (N = 27) to 387 microg/l (N = 16) for male C282Y homozygotes and from 357 microg/l (N = 42) to 69 microg/l (N = 12) for females, comparing those with no copies to those with one copy of rs884409. Functional testing of this CYBRD1 promoter polymorphism using a heterologous expression assay resulted in a 30% decrease in basal promoter activity relative to the common genotype (P = 0.004). This putative genetic modifier of iron overload expression accounts for 11% (95% CI 0.4%, 22.6%) of the variance in serum ferritin levels of C282Y homozygotes