Introduction Extracellular vesicles (EVs) from human Alzheimer's disease (AD) biospecimens contain amyloid beta (Aβ) peptide and tau. While AD EVs are known to affect brain disease pathobiology, their biochemical and molecular characterizations remain ill defined. Methods EVs were isolated from the cortical gray matter of 20 AD and 18 control brains. Tau and Aβ levels were measured by immunoassay. Differentially expressed EV proteins were assessed by quantitative proteomics and machine learning. Results Levels of pS396 tau and Aβ1–42 were significantly elevated in AD EVs. High levels of neuron‐ and glia‐specific factors are detected in control and AD EVs, respectively. Machine learning identified ANXA5, VGF, GPM6A, and ACTZ in AD EV compared to controls. They distinguished AD EVs from controls in the test sets with 88% accuracy. Discussion In addition to Aβ and tau, ANXA5, VGF, GPM6A, and ACTZ are new signature proteins in AD EVs.

Glycoproteomics is a powerful yet analytically challenging research tool. Software packages aiding the interpretation of complex glycopeptide tandem mass spectra have appeared, but their relative performance remains untested. Conducted through the HUPO Human Glycoproteomics Initiative, this community study, comprising both developers and users of glycoproteomics software, evaluates solutions for system-wide glycopeptide analysis. The same mass spectrometrybased glycoproteomics datasets from human serum were shared with participants and the relative team performance for N- and O-glycopeptide data analysis was comprehensively established by orthogonal performance tests. Although the results were variable, several high-performance glycoproteomics informatics strategies were identified. Deep analysis of the data revealed key performance-associated search parameters and led to recommendations for improved ‘high-coverage’ and ‘high-accuracy’ glycoproteomics search solutions. This study concludes that diverse software packages for comprehensive glycopeptide data analysis exist, points to several high-performance search strategies and specifies key variables that will guide future software developments and assist informatics decision-making in glycoproteomics.

previous studies on parkinson's disease mechanisms have shown dysregulated extracellular transport of α-synuclein and growth factors in the extracellular space. In the human brain these consist of perineuronal nets, interstitial matrices, and basement membranes, each composed of a set of collagens, non-collagenous glycoproteins, proteoglycans, and hyaluronan. The manner by which amyloidogenic proteins spread extracellularly, become seeded, oligomerize, and are taken up by cells, depends on intricate interactions with extracellular matrix molecules. We sought to assess the alterations to structure of glycosaminoglycans and proteins that occur in pD brain relative to controls of similar age. We found that PD differs markedly from normal brain in upregulation of extracellular matrix structural components including collagens, proteoglycans and glycosaminoglycan binding molecules. We also observed that levels of hemoglobin chains, possibly related to defects in iron metabolism, were enriched in PD brains. These findings shed important new light on disease processes that occur in association with pD. The volume of the extracellular space (~ 20%) that separates brain cell surfaces and through which molecules diffuse displays regional patterns that change during development, aging and neurodegeneration 1,2. The passage of protein molecules through the extracellular space depends on the geometries and chemical compositions of extracellular and cell surface molecular complexes, the specific binding domains thereof, and the fixed negative charges of glycosaminoglycan chains 3,4. Brain extracellular matrix (ECM) is composed of perineuronal nets (PNNs), interstitial matrices, and basement membranes (blood brain barrier), each consisting of a network of glycoproteins, proteoglycans, hyaluronan and collagens 5. Despite the obvious importance of the extracellular space to neural plasticity and neurodegeneration 6,7 , there is little information available on the alterations that occur to these molecules during Parkinson's disease (PD). Inflammation and disruption of the blood brain barrier can lead to infiltration of fibroblasts and trigger a fibrotic response in an attempt to restore normal function 8. Such fibrosis demolishes the structure of the ECM, and impedes healing by secreting inhibitory molecules and serves as a barrier to axons. Infiltration of fibroblasts leads to deposition of thrombin and fibrinogen and destruction of the integrity of the ECM. These inflammatory reactions lead to local neural degeneration and activation of glial cells. In PD, the activation of glial cells and recruitment of T-cells leads to increased pro-inflammatory cytokine release and increased levels of reactive oxygen and nitrogen species. While disruption is not believed to occur, activated microglia appear to induce blood brain barrier dysfunction in PD 9. Despite this, limited information is available concerning the changes in the distribution of ECM molecules in PD, with the exception of glycosaminoglycans (GAGs) found in senile plaques and...

Supplementary data are available at Bioinformatics online.

Myoglobins from horse heart muscle, horse skeletal muscle and sperm whale are widely used as calibration standards or test compounds for various mass spectrometric methodologies. In all such cases reported in the literature, a molecular weight value is used (16,950.5 and 17,199, respectively) which is based on the assumption that amino acid 122 in this 153 amino-acid-long protein is asparagine, overlooking a published suggestion that it is aspartic acid instead. Since the mass assignment accuracy for matrix-assisted laser desorption mass spectrometry is reported to be +/- 0.01% and for electrospray ionization +/- 0.0025%, and error of one mass unit in approximately 17,000 would be significant. The mass-to-charge ratio of ions of the tryptic peptide encompassing amino acid 122 derived from commercially available horse heart and horse skeletal myoglobins, the apomyoglobin of the latter, and the tryptic and chymotryptic peptide of sperm whale myoglobin proved that in both proteins amino acid 122 is indeed aspartic acid, rather than asparagine. This finding was further confirmed by the collision-induced dissociation spectra of the [M + H]+ ions of the tryptic peptides from the horse myoglobins and the chymotriptic peptide from sperm whale myoglobin. Thus, the correct molecular weight of horse myoglobin is 16,951.49 and that of the sperm whale protein is 17,199.91.

Human serum albumin was reacted with the (+)- and (-)-enantiomers of r-7,t-8-dihydroxy-t-9,t-10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene to determine if the chiral nature of the protein influences adduct formation. The alkylated proteins were analyzed directly by fluorescence line narrowing spectroscopy, and their spectra were compared to those of the model synthetic adducts N tau-(7,8,9-trihydroxy-r-7,t-8,t-9,c-10-tetrahydrobenzo[a]pyren-10- yl)histidine and 7,8,9-trihydroxy-r-7,t-8,t-9,c-10-tetrahydrobenzo[a]pyren- 10-yl N-t-BOC-alaninate ester. The results from these analyses indicated that different adducts were formed by the enantiomers of the diol epoxide. The adducted proteins were also enzymatically digested, and the 8,9-cis-dihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene-containing adducts and hydrolysis products were isolated by boronate affinity chromatography. Diode array UV, fast atom bombardment, and on-line atmospheric pressure ionization-mass spectral analysis of the HPLC purified products indicated that the more mutagenic and tumorigenic (+)-enantiomer forms carboxylic ester adducts with the protein at either Asp(187) or Glu(188), while the (-)-enantiomer forms N tau-histidine adducts at His(146). This previously unrealized enantiospecificity of the reaction of benzo[a]pyrene anti-diol epoxide with human serum albumin has important consequences for the application of the adducts as biomarkers of internal exposure.

Parkinson's disease (PD) is a neurological disorder characterized by the progressive loss of functional dopaminergic neurons in the nigrostriatal pathway in the brain. Although current treatments provide only symptomatic relief, gene therapy has the potential to slow or halt the degeneration of nigrostriatal dopamine neurons in PD patients. Adeno-associated viruses (AAV) are vectors of choice in gene therapy because of their well-characterized safety and efficacy profiles; however, although gene therapy has been successful in preclinical models of the disease, clinical trials in humans have failed to demonstrate efficacy. Significantly, all primary AAV receptors of the virus are glycans. We thus hypothesize that age related changes in glycan receptors of heparan sulfate (HS) proteoglycans (receptor for rAAV2), and/or -glycans with terminal galactose (receptor for rAAV9) results in poor adeno-associated virus binding in either the striatum or substantia nigra, or both, affecting transduction and gene delivery. To test our hypothesis we analyzed the striatum and substantia nigra for changes in HS,-glycans and proteomic signatures in young aged rat brain striatum and substantia nigra. We observed different brain region-specific HS disaccharide profiles in aged compared with young adult rats for brain region-specific profiles in striatum substantia nigra. We observed brain region- and age-specific -glycan compositional profiles with respect to the terminal galactose units that serve as receptors for AAV9. We also observed brain region-specific changes in protein expression in the aging nigrostriatal pathway. These studies provide insight into age- and brain region-specific changes in glycan receptors and proteome that will inform design of improved viral vectors for Parkinson Disease (PD) gene therapy.

Enteroviruses support cell-to-cell viral transmission prior to their canonical lytic spread of virus. Poliovirus (PV), a prototype for human pathogenic positive-sense RNA enteroviruses, and picornaviruses in general, transport multiple virions en bloc via infectious extracellular vesicles, 100~1000 nm in diameter, secreted from host cells. Using biochemical and biophysical methods we identify multiple components in secreted microvesicles, including mature PV virions; positive-sense genomic and negative-sense replicative, template viral RNA; essential viral replication proteins; and cellular proteins. Using cryo-electron tomography, we visualize the near-native three-dimensional architecture of secreted infectious microvesicles containing both virions and a unique morphological component that we describe as a mat-like structure. While the composition of these mat-like structures is not yet known, based on our biochemical data they are expected to be comprised of unencapsidated RNA and proteins. In addition to infectious microvesicles, CD9-positive exosomes released from PVinfected cells are also infectious and transport virions. Thus, our data show that, prior to cell lysis, nonenveloped viruses are secreted within infectious vesicles that also transport viral unencapsidated RNAs, viral and host proteins. Understanding the structure and function of these infectious particles helps elucidate the mechanism by which extracellular vesicles contribute to the spread of non-enveloped virus infection.