| 1. |
- Ayoglu, Burcu, et al.
(författare)
-
Affinity proteomics within rare diseases : a BIO-NMD study for blood biomarkers of muscular dystrophies
- 2014
-
Ingår i: EMBO Molecular Medicine. - : EMBO. - 1757-4676 .- 1757-4684. ; 6:7, s. 918-936
-
Tidskriftsartikel (refereegranskat)abstract
- Despite the recent progress in the broad-scaled analysis of proteins in body fluids, there is still a lack in protein profiling approaches for biomarkers of rare diseases. Scarcity of samples is the main obstacle hindering attempts to apply discovery driven protein profiling in rare diseases. We addressed this challenge by combining samples collected within the BIO-NMD consortium from four geographically dispersed clinical sites to identify protein markers associated with muscular dystrophy using an antibody bead array platform with 384 antibodies. Based on concordance in statistical significance and confirmatory results obtained from analysis of both serum and plasma, we identified eleven proteins associated with muscular dystrophy, among which four proteins were elevated in blood from muscular dystrophy patients: carbonic anhydrase III (CA3) and myosin light chain 3 (MYL3), both specifically expressed in slow-twitch muscle fibers and mitochondrial malate dehydrogenase 2 (MDH2) and electron transfer flavo-protein A (ETFA). Using age-matched sub-cohorts, 9 protein profiles correlating with disease progression and severity were identified, which hold promise for the development of new clinical tools for management of dystrophinopathies.
|
|
| 2. |
|
|
| 3. |
- Ayoglu, Burcu, et al.
(författare)
-
Anoctamin 2 identified as an autoimmune target in multiple sclerosis
- 2016
-
Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : National Academy of Sciences of the USA. - 0027-8424 .- 1091-6490. ; 113:8, s. 2188-2193
-
Tidskriftsartikel (refereegranskat)abstract
- Multiple sclerosis (MS) is the most common chronic inflammatory disease of the central nervous system and also is regarded as an autoimmune condition. However, the antigenic targets of the autoimmune response in MS have not yet been deciphered. In an effort to mine the autoantibody repertoire within MS, we profiled 2,169 plasma samples from MS cases and population-based controls using bead arrays built with 384 human protein fragments selected from an initial screening with 11,520 antigens. Our data revealed prominently increased autoantibody reactivity against the chloride-channel protein anoctamin 2 (ANO2) in MS cases compared with controls. This finding was corroborated in independent assays with alternative protein constructs and by epitope mapping with peptides covering the identified region of ANO2. Additionally, we found a strong interaction between the presence of ANO2 autoantibodies and the HLA complex MS-associated DRB1*15 allele, reinforcing a potential role for ANO2 autoreactivity in MS etiopathogenesis. Furthermore, immunofluorescence analysis in human MS brain tissue showed ANO2 expression as small cellular aggregates near and inside MS lesions. Thus this study represents one of the largest efforts to characterize the autoantibody repertoire within MS. The findings presented here demonstrate that an ANO2 autoimmune subphenotype may exist in MS and lay the groundwork for further studies focusing on the pathogenic role of ANO2 autoantibodies in MS.
|
|
| 4. |
- Ayoglu, Burcu, et al.
(författare)
-
Autoantibody profiling in multiple sclerosis using arrays of human protein fragments
- 2013
-
Ingår i: Molecular & Cellular Proteomics. - 1535-9476 .- 1535-9484. ; 12:9, s. 2657-2672
-
Tidskriftsartikel (refereegranskat)abstract
- Profiling the autoantibody repertoire with large antigen collections is emerging as a powerful tool for the identification of biomarkers for autoimmune diseases. Here, a systematic and undirected approach was taken to screen for profiles of IgG in human plasma from 90 individuals with multiple sclerosis related diagnoses. Reactivity pattern of 11,520 protein fragments (representing ̃38% of all human protein encoding genes) were generated on planar protein microarrays built within the Human Protein Atlas. For more than 2,000 antigens IgG reactivity was observed, among which 64% were found only in single individuals. We used reactivity distributions among multiple sclerosis subgroups to select 384 antigens, which were then reevaluated on planar microarrays, corroborated with suspension bead arrays in a larger cohort (n = 376) and confirmed for specificity in inhibition assays. Among the heterogeneous pattern within and across multiple sclerosis subtypes, differences in recognition frequencies were found for 51 antigens, which were enriched for proteins of transcriptional regulation. In conclusion, using protein fragments and complementary high-throughput protein array platforms facilitated an alternative route to discovery and verification of potentially disease-associated autoimmunity signatures, that are now proposed as additional antigens for large-scale validation studies across multiple sclerosis biobanks.
|
|
| 5. |
- Ayoglu, Burcu, et al.
(författare)
-
Multiplexed protein profiling by sequential affinity capture
- 2016
-
Ingår i: Proteomics. - : Wiley-Blackwell. - 1615-9853 .- 1615-9861. ; 16:8, s. 1251-1256
-
Tidskriftsartikel (refereegranskat)abstract
- Antibody microarrays enable parallelized and miniaturized analysis of clinical samples, and have proven to provide novel insights for the analysis of different proteomes. However, there are concerns that the performance of such direct labeling and single antibody assays are prone to off-target binding due to the sample context. To improve selectivity and sensitivity while maintaining the possibility to conduct multiplexed protein profiling, we developed a multiplexed and semi-automated sequential capture assay. This novel bead-based procedure encompasses a first antigen capture, labeling of captured protein targets on magnetic particles, combinatorial target elution and a read-out by a secondary capture bead array. We demonstrate in a proof-of-concept setting that target detection via two sequential affinity interactions reduced off-target contribution, while lowered background and noise levels, improved correlation to clinical values compared to single binder assays. We also compared sensitivity levels with single binder and classical sandwich assays, explored the possibility for DNA-based signal amplification, and demonstrate the applicability of the dual capture bead-based antibody microarray for biomarker analysis. Hence, the described concept enhances the possibilities for antibody array assays to be utilized for protein profiling in body fluids and beyond.
|
|
| 6. |
|
|
| 7. |
- Ayoglu, Burcu, et al.
(författare)
-
Systematic antibody and antigen-based proteomic profiling with microarrays
- 2011
-
Ingår i: EXPERT REVIEW OF MOLECULAR DIAGNOSTICS. - 1473-7159. ; 11:2, s. 219-234
-
Forskningsöversikt (refereegranskat)abstract
- Current approaches within affinity-based proteomics are driven both by the accessibility and availability of antigens and capture reagents, and by suitable multiplexed technologies onto which these are implemented. By combining planar microarrays and other multiparallel systems with sets of reagents, possibilities to discover new and unpredicted protein disease associations, either via directed hypothesis-driven or via undirected hypothesis-generating target selection, can be created. In the following stages, the discoveries made during these screening phases have to be verified for potential clinical relevance based on both technical and biological aspects. The use of affinity tools throughout discovery and verification has the potential to streamline the introduction of new markers, as transition into clinically required assay formats appears straightforward. In this article, we summarize some of the current building blocks within array-and affinity-based proteomic profiling with a focus on body fluids, by giving a perspective on how current and upcoming developments in this bioscience could enable a path of pursuit for biomarker discovery.
|
|
| 8. |
|
|
| 9. |
- Byström, Sanna, et al.
(författare)
-
Affinity Proteomic Profiling of Plasma, Cerebrospinal Fluid, and Brain Tissue within Multiple Sclerosis
- 2014
-
Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 13:11, s. 4607-4619
-
Tidskriftsartikel (refereegranskat)abstract
- The brain is a vital organ and because it is well shielded from the outside environment, possibilities for noninvasive analysis are often limited. Instead, fluids taken from the spinal cord or circulatory system are preferred sources for the discovery of candidate markers within neurological diseases. In the context of multiple sclerosis (MS), we applied an affinity proteomic strategy and screened 22 plasma samples with 4595 antibodies (3450 genes) on bead arrays, then defined 375 antibodies (334 genes) for targeted analysis in a set of 172 samples and finally used 101 antibodies (43 genes) on 443 plasma as well as 573 cerebrospinal spinal fluid (CSF) samples. This revealed alteration of protein profiles in relation to MS subtypes for IRF8, IL7, METTL14, SLC30A7, and GAP43. Respective antibodies were subsequently used for immunofluorescence on human post-mortem brain tissue with MS pathology for expression and association analysis. There, antibodies for IRF8, IL7, and METTL14 stained neurons in proximity of lesions, which highlighted these candidate protein targets for further studies within MS and brain tissue. The affinity proteomic translation of profiles discovered by profiling human body fluids and tissue provides a powerful strategy to suggest additional candidates to studies of neurological disorders.
|
|
| 10. |
- Häggmark, Anna, et al.
(författare)
-
Antibody-based profiling of cerebrospinal fluid within multiple sclerosis
- 2013
-
Ingår i: Proteomics. - : Wiley. - 1615-9853 .- 1615-9861. ; 13:15, s. 2256-2267
-
Tidskriftsartikel (refereegranskat)abstract
- Antibody suspension bead arrays have proven to enable multiplexed and high-throughput protein profiling in unfractionated plasma and serum samples through a direct labeling approach. We here describe the development and application of an assay for protein profiling of cerebrospinal fluid (CSF). While setting up the assay, systematic intensity differences between sample groups were observed that reflected inherent sample specific total protein amounts. Supplementing the labeling reaction with BSA and IgG diminished these differences without impairing the apparent sensitivity of the assay. We also assessed the effects of heat treatment on the analysis of CSF proteins and applied the assay to profile 43 selected proteins by 101 antibodies in 339 CSF samples from a multiple sclerosis (MS) cohort. Two proteins, GAP43 and SERPINA3 were found to have a discriminating potential with altered intensity levels between sample groups. GAP43 was detected at significantly lower levels in secondary progressive MS compared to early stages of MS and the control group of other neurological diseases. SERPINA3 instead was detected at higher levels in all MS patients compared to controls. The developed assay procedure now offers new possibilities for broad-scale protein profiling of CSF within neurological disorders.
|
|
