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AAbs provide value in identifying individuals at risk, stratifying patients with different clinical courses, improving our understanding of autoimmune destructions, identifying antigens for cellular immune response and providing candidates for prevention trials in T1D. A two-stage serological AAb screening against 6,000 human proteins was performed. A dual specificity tyrosine-phosphorylation-regulated kinase 2 (DYRK2) was validated with 36% sensitivity at 98% specificity by an orthogonal immunoassay. This is the first systematic screening for novel AAbs against large number of human proteins by protein arrays in T1D. A more comprehensive search for novel AAbs was performed using a knowledge-based approach by ELISA and a screening-based approach against 10,000 human proteins by NAPPA. Six AAbs were identified and validated with sensitivities ranged from 16% to 27% at 95% specificity. These two studies enriched the T1D “autoantigenome” and provided insights into T1D pathophysiology in an unprecedented breadth and width.
The rapid rise of T1D incidence suggests the potential involvement of environmental factors including viral infections. Sero-reactivity to 646 viral antigens was assessed in new-onset T1D patients. Antibody positive rate of EBV was significantly higher in cases than controls that suggested a potential role of EBV in T1D development. A high density-NAPPA platform was demonstrated with high reproducibility and sensitivity in profiling anti-viral antibodies.
This dissertation shows the power of a protein-array based immunoproteomics approach to characterize humoral immunoprofile against human and viral proteomes. The identification of novel T1D-specific AAbs and T1D-associated viruses will help to connect the nodes in T1D etiology and provide better understanding of T1D pathophysiology.
Human protein diversity arises as a result of alternative splicing, single nucleotide polymorphisms (SNPs) and posttranslational modifications. Because of these processes, each protein can exists as multiple variants in vivo. Tailored strategies are needed to study these protein variants and understand their role in health and disease. In this work we utilized quantitative mass spectrometric immunoassays to determine the protein variants concentration of beta-2-microglobulin, cystatin C, retinol binding protein, and transthyretin, in a population of 500 healthy individuals. Additionally, we determined the longitudinal concentration changes for the protein variants from four individuals over a 6 month period. Along with the native forms of the four proteins, 13 posttranslationally modified variants and 7 SNP-derived variants were detected and their concentration determined. Correlations of the variants concentration with geographical origin, gender, and age of the individuals were also examined. This work represents an important step toward building a catalog of protein variants concentrations and examining their longitudinal changes.
The apolipoprotein E (APOE) e4 allele is the most prevalent genetic risk factor for Alzheimer's disease (AD). Hippocampal volumes are generally smaller in AD patients carrying the e4 allele compared to e4 noncarriers. Here we examined the effect of APOE e4 on hippocampal morphometry in a large imaging database—the Alzheimer's Disease Neuroimaging Initiative (ADNI). We automatically segmented and constructed hippocampal surfaces from the baseline MR images of 725 subjects with known APOE genotype information including 167 with AD, 354 with mild cognitive impairment (MCI), and 204 normal controls. High-order correspondences between hippocampal surfaces were enforced across subjects with a novel inverse consistent surface fluid registration method. Multivariate statistics consisting of multivariate tensor-based morphometry (mTBM) and radial distance were computed for surface deformation analysis. Using Hotelling's T2 test, we found significant morphological deformation in APOE e4 carriers relative to noncarriers in the entire cohort as well as in the nondemented (pooled MCI and control) subjects, affecting the left hippocampus more than the right, and this effect was more pronounced in e4 homozygotes than heterozygotes. Our findings are consistent with previous studies that showed e4 carriers exhibit accelerated hippocampal atrophy; we extend these findings to a novel measure of hippocampal morphometry. Hippocampal morphometry has significant potential as an imaging biomarker of early stage AD.
Mild Cognitive Impairment (MCI) is a transitional stage between normal aging and dementia and people with MCI are at high risk of progression to dementia. MCI is attracting increasing attention, as it offers an opportunity to target the disease process during an early symptomatic stage. Structural magnetic resonance imaging (MRI) measures have been the mainstay of Alzheimer's disease (AD) imaging research, however, ventricular morphometry analysis remains challenging because of its complicated topological structure. Here we describe a novel ventricular morphometry system based on the hyperbolic Ricci flow method and tensor-based morphometry (TBM) statistics. Unlike prior ventricular surface parameterization methods, hyperbolic conformal parameterization is angle-preserving and does not have any singularities. Our system generates a one-to-one diffeomorphic mapping between ventricular surfaces with consistent boundary matching conditions. The TBM statistics encode a great deal of surface deformation information that could be inaccessible or overlooked by other methods. We applied our system to the baseline MRI scans of a set of MCI subjects from the Alzheimer's Disease Neuroimaging Initiative (ADNI: 71 MCI converters vs. 62 MCI stable). Although the combined ventricular area and volume features did not differ between the two groups, our fine-grained surface analysis revealed significant differences in the ventricular regions close to the temporal lobe and posterior cingulate, structures that are affected early in AD. Significant correlations were also detected between ventricular morphometry, neuropsychological measures, and a previously described imaging index based on fluorodeoxyglucose positron emission tomography (FDG-PET) scans. This novel ventricular morphometry method may offer a new and more sensitive approach to study preclinical and early symptomatic stage AD.
We sought to evaluate the reproducibility of a liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based approach to measure the stable-isotope enrichment of in vivo-labeled muscle ATP synthase β subunit (β-F1-ATPase), a protein most directly involved in ATP production, and whose abundance is reduced under a variety of circumstances. Muscle was obtained from a rat infused with stable-isotope-labeled leucine. The muscle was homogenized, β-F1-ATPase immunoprecipitated, and the protein was resolved using 1D-SDS PAGE. Following trypsin digestion of the isolated protein, the resultant peptide mixtures were subjected to analysis by HPLC-ESI-MS/MS, which resulted in the detection of multiple β-F1-ATPase peptides. There were three β-F1-ATPase unique peptides with a leucine residue in the amino acid sequence, and which were detected with high intensity relative to other peptides and assigned with >95% probability to β-F1-ATPase. These peptides were specifically targeted for fragmentation to access their stable-isotope enrichment based on MS/MS peak areas calculated from extracted ion chromatographs for selected labeled and unlabeled fragment ions. Results showed best linearity (R[superscript 2] = 0.99) in the detection of MS/MS peak areas for both labeled and unlabeled fragment ions, over a wide range of amounts of injected protein, specifically for the β-F1-ATPase[subscript 134-143] peptide. Measured stable-isotope enrichment was highly reproducible for the β-F1-ATPase[subscript 134-143] peptide (CV = 2.9%). Further, using mixtures of synthetic labeled and unlabeled peptides we determined that there is an excellent linear relationship (R[superscript 2] = 0.99) between measured and predicted enrichment for percent enrichments ranging between 0.009% and 8.185% for the β-F1-ATPase[subscript 134-143] peptide. The described approach provides a reliable approach to measure the stable-isotope enrichment of in-vivo-labeled muscle β-F1-ATPase based on the determination of the enrichment of the β-F1-ATPase[subscript 134-143] peptide.
Background: Healthy individuals on the lower end of the insulin sensitivity spectrum also have a reduced gene expression response to exercise for specific genes. The goal of this study was to determine the relationship between insulin sensitivity and exercise-induced gene expression in an unbiased, global manner.
Methods and Findings: Euglycemic clamps were used to measure insulin sensitivity and muscle biopsies were done at rest and 30 minutes after a single acute exercise bout in 14 healthy participants. Changes in mRNA expression were assessed using microarrays, and miRNA analysis was performed in a subset of 6 of the participants using sequencing techniques. Following exercise, 215 mRNAs were changed at the probe level (Bonferroni-corrected P<0.00000115). Pathway and Gene Ontology analysis showed enrichment in MAP kinase signaling, transcriptional regulation and DNA binding. Changes in several transcription factor mRNAs were correlated with insulin sensitivity, including MYC, r=0.71; SNF1LK, r=0.69; and ATF3, r= 0.61 (5 corrected for false discovery rate). Enrichment in the 5’-UTRs of exercise-responsive genes suggested regulation by common transcription factors, especially EGR1. miRNA species of interest that changed after exercise included miR-378, which is located in an intron of the PPARGC1B gene.
Conclusions: These results indicate that transcription factor gene expression responses to exercise depend highly on insulin sensitivity in healthy people. The overall pattern suggests a coordinated cycle by which exercise and insulin sensitivity regulate gene expression in muscle.
Proteins can exist as multiple proteoforms in vivo, as a result of alternative splicing and single-nucleotide polymorphisms (SNPs), as well as posttranslational processing. To address their clinical significance in a context of diagnostic information, proteoforms require a more in-depth analysis. Mass spectrometric immunoassays (MSIA) have been devised for studying structural diversity in human proteins. MSIA enables protein profiling in a simple and high-throughput manner, by combining the selectivity of targeted immunoassays, with the specificity of mass spectrometric detection. MSIA has been used for qualitative and quantitative analysis of single and multiple proteoforms, distinguishing between normal fluctuations and changes related to clinical conditions. This mini review offers an overview of the development and application of mass spectrometric immunoassays for clinical and population proteomics studies. Provided are examples of some recent developments, and also discussed are the trends and challenges in mass spectrometry-based immunoassays for the next-phase of clinical applications.