Deciphering ApoE Genotype-Driven Proteomic and Lipidomic Alterations in Alzheimer's Disease Across Distinct Brain Regions.

Alzheimer's disease (AD) is a neurodegenerative disease with a complex etiology influenced by confounding factors such as genetic polymorphisms, age, sex, and race. Traditionally, AD research has not prioritized these influences, resulting in dramatically skewed cohorts such as three times the number of Apolipoprotein E (APOE) ε4-allele carriers in AD relative to healthy cohorts. Thus, the resulting molecular changes in AD have previously been complicated by the influence of apolipoprotein E disparities. To explore how apolipoprotein E polymorphism influences AD progression, 62 post-mortem patients consisting of 33 AD and 29 controls (Ctrl) were studied to balance the number of ε4-allele carriers and facilitate a molecular comparison of the apolipoprotein E genotype. Lipid and protein perturbations were assessed across AD diagnosed brains compared to Ctrl brains, ε4 allele carriers (APOE4+ for those carrying 1 or 2 ε4s and APOE4- for non-ε4 carriers), and differences in ε3ε3 and ε3ε4 Ctrl brains across two brain regions (frontal cortex (FCX) and cerebellum (CBM)). The region-specific influences of apolipoprotein E on AD mechanisms showcased mitochondrial dysfunction and cell proteostasis at the core of AD pathophysiology in the post-mortem brains, indicating these two processes may be influenced by genotypic differences and brain morphology.

Quantitative proteomics of tau and Aß in detergent fractions from Alzheimer's disease brains.

The two hallmarks of Alzheimer's disease (AD) are amyloid-ß (Aß) plaques and neurofibrillary tangles marked by phosphorylated tau. Increasing evidence suggests that aggregating Aß drives tau accumulation, a process that involves synaptic degeneration leading to cognitive impairment. Conversely, there is a realization that non-fibrillar (oligomeric) forms of Aß mediate toxicity in AD. Fibrillar (filamentous) aggregates of proteins across the spectrum of the primary and secondary tauopathies were the focus of recent structural studies with a filament structure-based nosologic classification, but less emphasis was given to non-filamentous co-aggregates of insoluble proteins in the fractions derived from post-mortem human brains. Here, we revisited sarkosyl-soluble and -insoluble extracts to characterize tau and Aß species by quantitative targeted mass spectrometric proteomics, biochemical assays, and electron microscopy. AD brain sarkosyl-insoluble pellets were greatly enriched with Aß42 at almost equimolar levels to N-terminal truncated microtubule-binding region (MTBR) isoforms of tau with multiple site-specific post-translational modifications (PTMs). MTBR R3 and R4 tau peptides were most abundant in the sarkosyl-insoluble materials with a 10-fold higher concentration than N-terminal tau peptides. This indicates that the major proportion of the enriched tau was the aggregation-prone N-terminal and proline-rich region (PRR) of truncated mixed 4R and 3R tau with more 4R than 3R isoforms. High concentration and occupancies of site-specific phosphorylation pT181 (~22%) and pT217 (~16%) (key biomarkers of AD) along with other PTMs in the PRR and MTBR indicated a regional susceptibility of PTMs in aggregated tau. Immunogold labelling revealed that tau may exist in globular non-filamentous form (N-terminal intact tau) co-localized with Aß in the sarkosyl-insoluble pellets along with tau filaments (N-truncated MTBR tau). Our results suggest a model that Aß and tau interact forming globular aggregates, from which filamentous tau and Aß emerge. These characterizations contribute towards unravelling the sequence of events which lead to end-stage AD changes.

CSF ferritin in the clinicopathological progression of Alzheimer's disease and associations with APOE and inflammation biomarkers.

BACKGROUND: A putative role for iron in driving Alzheimer's disease (AD) progression is complicated by previously reported associations with neuroinflammation, apolipoprotein E and AD proteinopathy. To establish how iron interacts with clinicopathological features of AD and at what disease stage iron influences cognitive outcomes, we investigated the association of cerebrospinal fluid (CSF) biomarkers of iron (ferritin), inflammation (acute phase response proteins) and apolipoproteins with pathological biomarkers (CSF Aß42/t-tau, p-tau181), clinical staging and longitudinal cognitive deterioration in subjects from the BioFINDER cohort, with replication of key results in the Alzheimer's Disease Neuroimaging Initiative (ADNI) cohort. METHODS: Ferritin, acute phase response proteins (n=9) and apolipoproteins (n=6) were measured in CSF samples from BioFINDER (n=1239; 4 years cognitive follow-up) participants stratified by cognitive status (cognitively unimpaired, mild cognitive impairment, AD) and for the presence of amyloid and tangle pathology using CSF Aß42/t-tau (A+) and p-tau181 (T+). The ferritin and apolipoprotein E associations were replicated in the ADNI (n=264) cohort. RESULTS: In both cohorts, ferritin and apoE were elevated in A-T+ and A+T+ subjects (16%-40%), but not clinical diagnosis. Other apolipoproteins and acute phase response proteins increased with clinical diagnosis, not pathology. CSF ferritin was positively associated with p-tau181, which was mediated by apolipoprotein E. An optimised threshold of ferritin predicted cognitive deterioration in mild cognitive impairment subjects in the BioFINDER cohort, especially those people classified as A-T- and A+T-. CONCLUSIONS: CSF markers of iron and neuroinflammation have distinct associations with disease stages, while iron may be more intimately associated with apolipoprotein E and tau pathology.

Golgi-Dependent Copper Homeostasis Sustains Synaptic Development and Mitochondrial Content.

Rare genetic diseases preponderantly affect the nervous system causing neurodegeneration to neurodevelopmental disorders. This is the case for both Menkes and Wilson disease, arising from mutations in ATP7A and ATP7B, respectively. The ATP7A and ATP7B proteins localize to the Golgi and regulate copper homeostasis. We demonstrate genetic and biochemical interactions between ATP7 paralogs with the conserved oligomeric Golgi (COG) complex, a Golgi apparatus vesicular tether. Disruption of Drosophila copper homeostasis by ATP7 tissue-specific transgenic expression caused alterations in epidermis, aminergic, sensory, and motor neurons. Prominent among neuronal phenotypes was a decreased mitochondrial content at synapses, a phenotype that paralleled with alterations of synaptic morphology, transmission, and plasticity. These neuronal and synaptic phenotypes caused by transgenic expression of ATP7 were rescued by downregulation of COG complex subunits. We conclude that the integrity of Golgi-dependent copper homeostasis mechanisms, requiring ATP7 and COG, are necessary to maintain mitochondria functional integrity and localization to synapses.SIGNIFICANCE STATEMENT Menkes and Wilson disease affect copper homeostasis and characteristically afflict the nervous system. However, their molecular neuropathology mechanisms remain mostly unexplored. We demonstrate that copper homeostasis in neurons is maintained by two factors that localize to the Golgi apparatus, ATP7 and the conserved oligomeric Golgi (COG) complex. Disruption of these mechanisms affect mitochondrial function and localization to synapses as well as neurotransmission and synaptic plasticity. These findings suggest communication between the Golgi apparatus and mitochondria through homeostatically controlled cellular copper levels and copper-dependent enzymatic activities in both organelles.

GH18 endo-ß-N-acetylglucosaminidases use distinct mechanisms to process hybrid-type N-linked glycans.

N-glycosylation is one of the most abundant posttranslational modifications of proteins, essential for many physiological processes, including protein folding, protein stability, oligomerization and aggregation, and molecular recognition events. Defects in the N-glycosylation pathway cause diseases that are classified as congenital disorders of glycosylation. The ability to manipulate protein N-glycosylation is critical not only to our fundamental understanding of biology but also for the development of new drugs for a wide range of human diseases. Chemoenzymatic synthesis using engineered endo-ß-N-acetylglucosaminidases (ENGases) has been used extensively to modulate the chemistry of N-glycosylated proteins. However, defining the molecular mechanisms by which ENGases specifically recognize and process N-glycans remains a major challenge. Here we present the X-ray crystal structure of the ENGase EndoBT-3987 from Bacteroides thetaiotaomicron in complex with a hybrid-type glycan product. In combination with alanine scanning mutagenesis, molecular docking calculations and enzymatic activity measurements conducted on a chemically engineered monoclonal antibody substrate unveil two mechanisms for hybrid-type recognition and processing by paradigmatic ENGases. Altogether, the experimental data provide pivotal insight into the molecular mechanism of substrate recognition and specificity for GH18 ENGases and further advance our understanding of chemoenzymatic synthesis and remodeling of homogeneous N-glycan glycoproteins.

The Neuroinflammatory Acute Phase Response in Parkinsonian-Related Disorders.

BACKGROUND: Neuroinflammation is implicated in the pathophysiology of Parkinson's disease (PD) and related conditions, yet prior clinical biomarker data report mixed findings. OBJECTIVES: The aim was to measure a panel of neuroinflammatory acute phase response (APR) proteins in the cerebrospinal fluid (CSF) of participants with PD and related disorders. METHODS: Eleven APR proteins were measured in the CSF of 867 participants from the BioFINDER cohort who were healthy (612) or had a diagnosis of PD (155), multiple system atrophy (MSA) (26), progressive supranuclear palsy (PSP) (22), dementia with Lewy bodies (DLB) (23), or Parkinson's disease with dementia (PDD) (29). RESULTS: CSF APR proteins were mostly unchanged in PD, with only haptoglobin and α1-antitrypsin significantly elevated compared to controls. These proteins were variably increased in the other disorders. Certain protein components yielded unique signatures according to diagnosis: ferritin and transthyretin were selectively elevated in MSA and discriminated these patients from all others. Haptoglobin was selectively increased in PSP, discriminating this disease from MSA when used in combination with ferritin and transthyretin. This panel of proteins did not correlate well with severity of motor impairment in any disease category, but several (particularly ceruloplasmin and ferritin) were associated with memory performance (Mini-Mental State Examination) in patients with DLB and PDD. CONCLUSIONS: These findings provide new insights into inflammatory changes in PD and related disorders while also introducing biomarkers of potential clinical diagnostic utility. © 2022 International Parkinson and Movement Disorder Society.

Analysis of plasma proteins using 2D gels and novel fluorescent probes: in search of blood based biomarkers for Alzheimer's disease.

BACKGROUND: The Australian Imaging and Biomarker Lifestyle (AIBL) study of aging is designed to aid the discovery of biomarkers. The current study aimed to discover differentially expressed plasma proteins that could yield a blood-based screening tool for Alzheimer's disease. METHODS: The concentration of proteins in plasma covers a vast range of 12 orders of magnitude. Therefore, to search for medium to low abundant biomarkers and elucidate mechanisms of AD, we immuno-depleted the most abundant plasma proteins and pre-fractionated the remaining proteins by HPLC, prior to two-dimensional gel electrophoresis. The relative levels of approximately 3400 protein species resolved on the 2D gels were compared using in-gel differential analysis with spectrally resolved fluorescent protein detection dyes (Zdyes™). Here we report on analysis of pooled plasma samples from an initial screen of a sex-matched cohort of 72 probable AD patients and 72 healthy controls from the baseline time point of AIBL. RESULTS: We report significant changes in variants of apolipoprotein E, haptoglobin, α1 anti-trypsin, inter-α trypsin inhibitor, histidine-rich glycoprotein, and a protein of unknown identity. α1 anti-trypsin and α1 anti-chymotrypsin demonstrated plasma concentrations that were dependent on APOE ε4 allele dose. Our analysis also identified an association with the level of Vitamin D binding protein fragments and complement factor I with sex. We then conducted a preliminary validation study, on unique individual samples compared to the discovery cohort, using a targeted LC-MS/MS assay on a subset of discovered biomarkers. We found that targets that displayed a high degree of isoform specific changes in the 2D gels were not changed in the targeted MS assay which reports on the total level of the biomarker. CONCLUSIONS: This demonstrates that further development of mass spectrometry assays is needed to capture the isoform complexity that exists in theses biological samples. However, this study indicates that a peripheral protein signature has potential to aid in the characterization of AD.

Enhanced ion mobility resolution of Abeta isomers from human brain using high-resolution demultiplexing software.

Isomerization of aspartic acid (Asp) residues in long-lived proteins is a key feature associated with neurodegenerative proteinopathies such as Alzheimer's disease (AD). Recently, using ultra high-performance liquid chromatography (UHPLC) coupled with drift tube ion mobility mass spectrometry (DTIMS-MS), we documented the extensive Asp isomerization in amyloid-beta (Aß) peptides depositing in the extracellular cortical plaques (senile plaques) of the AD brain. Aß1-15 was estimated to be ~ 85% isomerized, while Aß4-15 another major constituent of these senile plaques was ~ 50% isomerized in AD brain. Low resolution on the standard demultiplexed ion mobility resulted in poor separation of these N-truncated Aß isomers in the ion mobility domain. Here, using the same ion multiplexed dataset, we applied new post-acquisition data reconstruction technique, high-resolution demultiplexing (HRdm), to improve the resolution of these Aß isomers in the ion mobility dimension. We demonstrate that for the complex proteomic AD brain digests, HRdm could successfully resolve three out of four major Asp isomers of Aß1-15. For Aß2-15 and Aß4-15, the significant resolution enhancement in the HRdm data resulted in baseline peak separation of the respective Asp isomers. An analysis of two-peak resolution (Rpp) and peak-to-peak separation (ΔP) indicated twofold enhancement for the Asp-isomerized Aß species. HRdm performed with an effective resolving power (Rp) of between 150 and 160 for the highest deconvolution settings in comparison to ~ 40 to 65 in the standard settings. These major resolution improvements in the ion mobility domain for the endogenous Aß isomers demonstrate the feasibility of in situ measurement of peptide isomers and their role in the mechanism of amyloid plaque formation in AD.

Increased glutaminyl cyclase activity in brains of Alzheimer's disease individuals.

Glutaminyl cyclases (QC) catalyze the formation of neurotoxic pGlu-modified amyloid-ß peptides found in the brains of people with Alzheimer's disease (AD). Reports of several-fold increases in soluble QC (sQC) expression in the brain and peripheral circulation of AD individuals has prompted the development of QC inhibitors as potential AD therapeutics. There is, however, a lack of standardized quantitative data on QC expression in human tissues, precluding inter-laboratory comparison and validation. We tested the hypothesis that QC is elevated in AD tissues by quantifying levels of sQC protein and activity in post-mortem brain tissues from AD and age-matched control individuals. We found a modest but statistically significant increase in sQC protein, which paralleled a similar increase in enzyme activity. In plasma samples sourced from the Australian Imaging, Biomarker and Lifestyle study we determined that QC activity was not different between the AD and control group, though a modest increase was observed in female AD individuals compared to controls. Plasma QC activity was further correlated with levels of circulating monocytes in AD individuals. These data provide quantitative evidence that alterations in QC expression are associated with AD pathology.

Early existence and biochemical evolution characterise acutely synaptotoxic PrPSc.

Although considerable evidence supports that misfolded prion protein (PrPSc) is the principal component of "prions", underpinning both transmissibility and neurotoxicity, clear consensus around a number of fundamental aspects of pathogenesis has not been achieved, including the time of appearance of neurotoxic species during disease evolution. Utilizing a recently reported electrophysiology paradigm, we assessed the acute synaptotoxicity of ex vivo PrPSc prepared as crude homogenates from brains of M1000 infected wild-type mice (cM1000) harvested at time-points representing 30%, 50%, 70% and 100% of the terminal stage of disease (TSD). Acute synaptotoxicity was assessed by measuring the capacity of cM1000 to impair hippocampal CA1 region long-term potentiation (LTP) and post-tetanic potentiation (PTP) in explant slices. Of particular note, cM1000 from 30% of the TSD was able to cause significant impairment of LTP and PTP, with the induced failure of LTP increasing over subsequent time-points while the capacity of cM1000 to induce PTP failure appeared maximal even at this early stage of disease progression. Evidence that the synaptotoxicity directly related to PrP species was demonstrated by the significant rescue of LTP dysfunction at each time-point through immuno-depletion of >50% of total PrP species from cM1000 preparations. Moreover, similar to our previous observations at the terminal stage of M1000 prion disease, size fractionation chromatography revealed that capacity for acute synpatotoxicity correlated with predominance of oligomeric PrP species in infected brains across all time points, with the profile appearing maximised by 50% of the TSD. Using enhanced sensitivity western blotting, modestly proteinase K (PK)-resistant PrPSc was detectable at very low levels in cM1000 at 30% of the TSD, becoming robustly detectable by 70% of the TSD at which time substantial levels of highly PK-resistant PrPSc was also evident. Further illustrating the biochemical evolution of acutely synaptotoxic species the synaptotoxicity of cM1000 from 30%, 50% and 70% of the TSD, but not at 100% TSD, was abolished by digestion of immuno-captured PrP species with mild PK treatment (5µg/ml for an hour at 37°C), demonstrating that the predominant synaptotoxic PrPSc species up to and including 70% of the TSD were proteinase-sensitive. Overall, these findings in combination with our previous assessments of transmitting prions support that synaptotoxic and infectious M1000 PrPSc species co-exist from at least 30% of the TSD, simultaneously increasing thereafter, albeit with eventual plateauing of transmitting conformers.

Quantification of metallothionein-III in brain tissues using liquid chromatography tandem mass spectrometry.

Metallothioneins (MTs) are crucial for metal ion homeostasis in mammalian cells. Specialized mass spectrometry methods have been developed to detect MTs in tissue extracts, though facile methods with scalable throughput are lacking. To improve analytical throughput and repeatability, we developed a standardised liquid chromatography tandem mass spectrometry (LC-MS/MS) method for robust determination of metallothionein-3 (MT3) that is amenable to microplate processing. This method uses standard protein digestion conditions with commercially available reagents and commonly practiced reversed-phase chromatography, detecting MT3 at low ng/mL levels in human brain tissue extracts. We found that trypsin digestion largely underestimated MT3 levels, whereas endopeptidase Lys-C yielded vastly higher signals with low replicate variance. The choice of target peptide was critical for accurate MT3 detection - a peptide in the α-domain yielded the most robust signals. We demonstrate the utility of this method by comparing the expression of MT3 in post-mortem brain tissues of a cohort of Alzheimer's disease (AD) individuals and age-matched controls.

PrPSc Oligomerization Appears Dynamic, Quickly Engendering Inherent M1000 Acute Synaptotoxicity.

Prion diseases are neurodegenerative disorders pathogenically linked to cellular prion protein (PrPC) misfolding into abnormal conformers (PrPSc), with PrPSc underpinning both transmission and synaptotoxicity. Although the biophysical features of PrPSc required to induce acute synaptic dysfunction remain incompletely defined, we recently reported that acutely synaptotoxic PrPSc appeared to be oligomeric. We herein provide further insights into the kinetic and requisite biophysical characteristics of acutely synaptotoxic ex vivo PrPSc derived from the brains of mice dying from M1000 prion disease. Pooled fractions of M1000 PrPSc located within the molecular weight range approximating monomeric PrP (mM1000) generated through size exclusion chromatography were found to harbor acute synaptotoxicity equivalent to preformed oligomeric fractions (oM1000). Subsequent investigation showed mM1000 corresponded to PrPSc rapidly concatenating in physiological buffer to exist as predominantly, closely associated, small oligomers. The oligomerization of PrP in mM1000 could be substantially mitigated by treatment with the antiaggregation compound epigallocatechin gallate, thereby maintaining the PrPSc as primarily nonoligomeric with completely abrogated acute synaptotoxicity; moreover, despite epigallocatechin gallate treatment, pooled oM1000 remained oligomeric and acutely synaptotoxic. A similar tendency to rapid formation of oligomers was observed for PrPC when monomeric fractions derived from size exclusion chromatography of normal brain homogenates (mNBH) were pooled, but neither mNBH nor preformed higher-order NBH complexes (oNBH) were acutely synaptotoxic. Oligomers formed from mNBH could be reduced to mainly monomers (<100 kDa) after enzymatic digestion of nucleic acids, whereas higher-order PrP assemblies derived from pooled mM1000, oM1000, and oNBH resisted such treatment. Collectively, these findings support that oligomerization of PrPSc into small multimeric assemblies appears to be a critical biophysical feature for engendering inherent acute synaptotoxicity, with preformed oligomers found in oM1000 appearing to be stable, tightly self-associated ensembles that coexist in dynamic equilibrium with mM1000, with the latter appearing capable of rapid aggregation, albeit initially forming smaller, weakly self-associated, acutely synaptotoxic oligomers.

In situ 3D visualization of biomineralization matrix proteins.

Calcium biominerals occur in all major animal phyla, and through biomolecular control, exhibit such diverse structures as exoskeletons, shells, bones, teeth and earstones (otoliths). Determining the three-dimensional expression of key biomineral proteins, however, has proven challenging as typical protein identification methods either lose spatial resolution during dissolution of the mineral phase or are costly and limited to two-dimensional expression of high abundance proteins. Here we present a modification of the CLARITY and ACT-PRESTO protocols to visualize and confirm, for the first time, the timing of expression and function of two key regulators of biomineralization.

Prion acute synaptotoxicity is largely driven by protease-resistant PrPSc species.

Although misfolding of normal prion protein (PrPC) into abnormal conformers (PrPSc) is critical for prion disease pathogenesis our current understanding of the underlying molecular pathophysiology is rudimentary. Exploiting an electrophysiology paradigm, herein we report that at least modestly proteinase K (PK)-resistant PrPSc (PrPres) species are acutely synaptotoxic. Brief exposure to ex vivo PrPSc from two mouse-adapted prion strains (M1000 and MU02) prepared as crude brain homogenates (cM1000 and cMU02) and cell lysates from chronically M1000-infected RK13 cells (MoRK13-Inf) caused significant impairment of hippocampal CA1 region long-term potentiation (LTP), with the LTP disruption approximating that reported during the evolution of murine prion disease. Proof of PrPSc (especially PrPres) species as the synaptotoxic agent was demonstrated by: significant rescue of LTP following selective immuno-depletion of total PrP from cM1000 (dM1000); modestly PK-treated cM1000 (PK+M1000) retaining full synaptotoxicity; and restoration of the LTP impairment when employing reconstituted, PK-eluted, immuno-precipitated M1000 preparations (PK+IP-M1000). Additional detailed electrophysiological analyses exemplified by impairment of post-tetanic potentiation (PTP) suggest possible heightened pre-synaptic vulnerability to the acute synaptotoxicity. This dysfunction correlated with cumulative insufficiency of replenishment of the readily releasable pool (RRP) of vesicles during repeated high-frequency stimulation utilised for induction of LTP. Broadly comparable results with LTP and PTP impairment were obtained utilizing hippocampal slices from PrPC knockout (PrPo/o) mice, with cM1000 serial dilution assessments revealing similar sensitivity of PrPo/o and wild type (WT) slices. Size fractionation chromatography demonstrated that synaptotoxic PrP correlated with PK-resistant species >100kDa, consistent with multimeric PrPSc, with levels of these species >6 ng/ml appearing sufficient to induce synaptic dysfunction. Biochemical analyses of hippocampal slices manifesting acute synaptotoxicity demonstrated reduced levels of multiple key synaptic proteins, albeit with noteworthy differences in PrPo/o slices, while such changes were absent in hippocampi demonstrating rescued LTP through treatment with dM1000. Our findings offer important new mechanistic insights into the synaptic impairment underlying prion disease, enhancing prospects for development of targeted effective therapies.

Optimizing red blood cell protein extraction for biomarker quantitation with mass spectrometry.

Red blood cells (RBC) are the most common cell type found in blood. They might serve as reservoir for biomarker research as they are anuclear and lack the ability to synthesize proteins. Not many biomarker assays, however, have been conducted on RBC because of their large dynamic range of proteins, high abundance of lipids, and hemoglobin interferences. Here, we developed a semiquantitative mass spectrometry-based assay that targeted 144 proteins and compared the efficiency of urea, sodium deoxycholate, acetonitrile, and HemoVoid™ in their extraction of the RBC proteome. Our results indicate that protein extraction with HemoVoid™ led to hemoglobin reduction and increased detection of low abundance proteins. Although hemoglobin interference after deoxycholate and urea extraction was high, there were adequate amounts of low abundance proteins for quantitation. Extraction with acetonitrile led to an overall decrease in protein abundances probably as a result of precipitation. Overall, the best compromise in sensitivity and sample processing time was achieved with the urea-trypsin digestion protocol. This provided the basis for large-scale evaluations of protein targets as potential blood-based biomarkers. As a proof of concept, we applied this assay to determine that alpha-synuclein, a prominent marker in Parkinson's disease, has an average concentration of approximately 40 µg mL-1 in RBC. This is important to know as the concentration of alpha-synuclein in plasma, typically in the picogram per milliliter range, might be partially derived from lysed RBC. Utilization of this assay will prove useful for future biomarker studies and provide a more complete analytical toolbox for the measurement of blood-derived proteins. Graphical abstract.

Zinc Transporter-3 Knockout Mice Demonstrate Age-Dependent Alterations in the Metalloproteome.

Metals are critical cellular elements that are involved in a variety of cellular processes, with recent literature demonstrating that zinc, and the synaptic zinc transporter (ZnT3), are specifically involved in learning and memory and may also be key players in age-related neurodegenerative disorders such as Alzheimer's disease. Whilst the cellular content and location of metals is critical, recent data has demonstrated that the metalation state of proteins is a determinant of protein function and potential toxicity. As we have previously reported that ZnT3 knockout (KO) mice have deficits in total zinc levels at both 3 and 6 months of age, we were interested in whether there might be changes in the metalloproteomic profile in these animals. To do this, we utilised size exclusion chromatography-inductively coupled plasma mass spectrometry (SEC-ICP-MS) and examined hippocampal homogenates from ZnT3 KO and age-matched wild-type mice at 3, 6 and 18 months of age. Our data suggest that there are alterations in specific metal binding proteins, for zinc, copper and iron all being modulated in the ZnT3 KO mice compared to wild-type (WT). These data suggest that ZnT3 KO mice may have impairments in the levels or localisation of multiple transition metals, and that copper- and iron-dependent cellular pathways may also be impacted in these mice.

Establishing Signature Fragments for Identification and Sequencing of Dityrosine Cross-Linked Peptides Using Ultraviolet Photodissociation Mass Spectrometry.

Dityrosine cross-linking of Aß peptides and α-synuclein is increasingly becoming recognized as a biomarker of neuropathological diseases. However, there remains a need for the development of analytical methods that enable the specific and selective identification of dityrosine cross-linked proteins and peptides in complex biological samples. Here, we report that the gas-phase fragmentation of protonated dityrosine cross-linked peptides under ultraviolet photodissociation (UVPD) tandem mass spectrometry (MS/MS) conditions results in the cleavage across Cα and Cß atoms of the dityrosine residue. This Cα-Cß cleavage in UVPD-MS/MS results in the formation of diagnostic pairs of product ions, providing information on the two individual peptides involved in the cross-linking, resolving the intrinsic "n2 problem" plaguing the identification of this post-translational modification (PTM) by tandem mass spectrometry. Sequencing of a heterodimeric dityrosine cross-linked peptide was demonstrated using hybrid UVPD-MS/MS and CID-MS3 on a diagnostic pair of product ions. In combination with dedicated MS-cleavable MSn software, UVPD-MSn therefore provides an avenue to selectively discover and describe dityrosine cross-linked peptides. Additionally, observation of dityrosine-specific "reporter ions" at m/z 240.1019 and m/z 223.0752 in UVPD-MS/MS will be useful for the validation of the dityrosine cross-linked peptides.

Biochemically-defined pools of amyloid-ß in sporadic Alzheimer's disease: correlation with amyloid PET.

We fractionated frontal cortical grey matter from human Alzheimer's disease and control subjects into four biochemically defined pools that represent four distinct compartments: soluble/cytosolic, peripheral membrane/vesicular cargo, integral lipid/membranous pools and aggregated/insoluble debris. Most of the readily extractable amyloid-ß remains associated with a lipid/membranous compartment. There is an exchange of amyloid-ß between the biochemical pools that was lost for the amyloid-ß42 species in Alzheimer's disease, consistent with the peptide being irreversibly trapped in extracellular deposits. The quantitative amyloid-ß data, combined with magnetic resonance imaging volumetric analysis of the amount of cortical grey matter in brain, allowed us to estimate the total mass of amyloid-ß in Alzheimer's disease (6.5 mg) and control (1.7 mg) brains. The threshold positron emission tomography standard uptake value ratio of 1.4 equates to 5.0 µg amyloid-ß/g of grey matter and the mean Alzheimer's disease dementia standard uptake value ratio level of 2.3 equates to 11.20 µg amyloid-ß/g of grey matter. It takes 19 years to accumulate amyloid from the threshold positron emission tomography standard uptake value ratio to the mean value observed for Alzheimer's disease dementia. This accumulation time window combined with the difference of 4.8 mg of amyloid-ß between Alzheimer's disease and control brain allows for a first approximation of amyloid-ß accumulation of 28 ng/h. This equates to an estimated 2-5% of the total amyloid-ß production being deposited as insoluble plaques. Understanding these rates of amyloid-ß accumulation allows for a more quantitative approach in targeting the failure of amyloid-ß clearance in sporadic Alzheimer's disease.

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 Aß(1-16) using ESI tandem mass spectrometry. We then detailed the fragmentation pattern of dityrosine cross-linked Aß(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 Aß 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.