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Neuroproteomic profiling of human body fluids
KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.ORCID iD: 0000-0002-0056-1313
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

This thesis provides results from affinity based studies where human body fluids were profiled to find markers for neurological diseases. Both proteins and autoantibodies were analysed using microarray technologies that can profile hundreds of analytes and hundreds of samples in parallel using small sample volumes. A central element in this work was to develop and apply new methods to study cerebrospinal fluid (CSF), which is the fluid in direct contact with the brain. CSF contains proteins reflecting the physiological state of the central nervous system and therefore offers a unique insight into proteins associated to neurological disorders. As a complement to CSF, bloodderived samples such as serum and plasma, were also investigated as these represent potential sources of disease related proteins. The work presented here summarises the development of assay protocols to study protein and autoantibodies in CSF and blood using planar and bead-based microarrays.

In Paper I, an antibody-based protocol was developed to enable multiplexed protein profiling in CSF. The protocol was then applied for a first analysis within multiple sclerosis (MS) patients. In Paper II, the results were further evaluated in additional CSF as well as plasma samples. Based on the CSF analysis we found two proteins associated to MS; GAP43, a protein related to disease progression and SERPINA3, a protein involved in inflammation. In addition, four other proteins; IRF8, METTL14, IL7 and SLC30A7, were found to have altered plasma levels between the patient groups. The expression of these proteins were further investigated by immunofluorescent staining of human brain tissue, revealing differential localisation of proteins in diseased and healthy brain. In Paper III, a study on extensive protein profiling of plasma in the context of another neurodegenerative disorder, amyotrophic lateral sclerosis (ALS), is described. The levels of three proteins, namely NEFM, RGS18 and SCL25A20, were found to be elevated in ALS patients compared to controls. Among these, NEFM also indicated association to disease subtype as the levels were elevated in patients with definite compared to suspected diagnosis.

In addition to antibodies, we also utilised antigens on microarrays to screen for the presence of autoantibodies in body fluids. In Paper IV, a strategy for this analysis was developed using protein fragments and two types of microarrays. This strategy was then applied for profiling of the autoantibody repertoire of MS patients, revealing 51 protein fragments with potential disease relevance. Interestingly, comparison of plasma and CSF samples obtained from the same patients indicated high concordance of antibodies between the two body fluids. In Paper V, a similar strategy was applied to narcolepsy, another neurological disorder. Our investigation of antibodies in serum revealed higher reactivity towards METTL22, NT5C1A and TMEM134 compared to controls in two independent sample materials.

In conclusion, the presented work constitutes a framework of proteomic assays for enhanced exploration of proteins and autoantibodies in neuroscience. Moreover, we have reported identification of several potential disease markers to be further investigated within neurological disorders.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2015. , viii, 67 p.
Series
TRITA-BIO-Report, ISSN 1654-2312 ; 2015:2
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Research subject
Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-158944ISBN: 978-91-7595-402-8 (print)OAI: oai:DiVA.org:kth-158944DiVA: diva2:780973
Public defence
2015-02-06, Rockefellersalen, KI, Solna, 09:00 (English)
Opponent
Supervisors
Note

QC 20150116

Available from: 2015-01-16 Created: 2015-01-15 Last updated: 2015-01-16Bibliographically approved
List of papers
1. Antibody-based profiling of cerebrospinal fluid within multiple sclerosis
Open this publication in new window or tab >>Antibody-based profiling of cerebrospinal fluid within multiple sclerosis
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2013 (English)In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 13, no 15, 2256-2267 p.Article in journal (Refereed) Published
Abstract [en]

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.

Keyword
Antibody microarrays, Biomarker discovery, Cerebrospinal fluid, Multiplexed proteomics technology, Protein arrays, Proteome profiling
National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-134062 (URN)10.1002/pmic.201200580 (DOI)000327008300007 ()2-s2.0-84881230169 (Scopus ID)
Funder
Science for Life Laboratory - a national resource center for high-throughput molecular bioscienceVinnovaKnut and Alice Wallenberg Foundation
Note

QC 20131115

Available from: 2013-11-15 Created: 2013-11-15 Last updated: 2017-12-06Bibliographically approved
2. Affinity Proteomic Profiling of Plasma, Cerebrospinal Fluid, and Brain Tissue within Multiple Sclerosis
Open this publication in new window or tab >>Affinity Proteomic Profiling of Plasma, Cerebrospinal Fluid, and Brain Tissue within Multiple Sclerosis
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2014 (English)In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 13, no 11, 4607-4619 p.Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
American Chemical Society (ACS), 2014
Keyword
antibodies, suspension bead arrays, plasma, CSF, brain tissue, immunofluorescence, multiple sclerosis
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-150383 (URN)10.1021/pr500609e (DOI)000344636500012 ()25231264 (PubMedID)2-s2.0-84908890596 (Scopus ID)
Funder
Swedish Research CouncilAFA InsuranceScience for Life Laboratory - a national resource center for high-throughput molecular bioscienceKnut and Alice Wallenberg Foundation
Note

QC 20141212

Updated from manuscript to article in journal.

Available from: 2014-09-02 Created: 2014-09-02 Last updated: 2017-12-05Bibliographically approved
3. Plasma profiling revelas three proteins associated to amyotrophic lateral sclerosis
Open this publication in new window or tab >>Plasma profiling revelas three proteins associated to amyotrophic lateral sclerosis
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2014 (English)In: Annals of Clinical and Translational Neurology, ISSN 2328-9503, Vol. 1, no 8, 544-553 p.Article in journal (Refereed) Published
Abstract [en]

OBJECTIVE: Amyotrophic lateral sclerosis (ALS) is the most common adult motor neuron disease leading to muscular paralysis and death within 3-5 years from onset. Currently, there are no reliable and sensitive markers able to substantially shorten the diagnosis delay. The objective of the study was to analyze a large number of proteins in plasma from patients with various clinical phenotypes of ALS in search for novel proteins or protein profiles that could serve as potential indicators of disease.

METHODS: Affinity proteomics in the form of antibody suspension bead arrays were applied to profile plasma samples from 367 ALS patients and 101 controls. The plasma protein content was directly labeled and protein profiles obtained using 352 antibodies from the Human Protein Atlas targeting 278 proteins. A focused bead array was then built to further profile eight selected protein targets in all available samples.

RESULTS: Disease-associated significant differences were observed and replicated for profiles from antibodies targeting the proteins: neurofilament medium polypeptide (NEFM), solute carrier family 25 (SLC25A20), and regulator of G-protein signaling 18 (RGS18).

INTERPRETATION: Upon further validation in several independent cohorts with inclusion of a broad range of other neurological disorders as controls, the alterations of these three protein profiles in plasma could potentially provide new molecular markers of disease that contribute to the quest of understanding ALS pathology.

National Category
Biomedical Laboratory Science/Technology
Identifiers
urn:nbn:se:kth:diva-158941 (URN)10.1002/acn3.83 (DOI)25356426 (PubMedID)
Note

QC 20150115

Available from: 2015-01-15 Created: 2015-01-15 Last updated: 2015-01-16Bibliographically approved
4. Autoantibody profiling in multiple sclerosis using arrays of human protein fragments
Open this publication in new window or tab >>Autoantibody profiling in multiple sclerosis using arrays of human protein fragments
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2013 (English)In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 12, no 9, 2657-2672 p.Article in journal (Refereed) Published
Abstract [en]

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.

Keyword
autoantibody, immunoglobulin G, adult, aged, article, controlled study, female, human, major clinical study, male, multiple sclerosis, priority journal, protein microarray, transcription regulation
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-133813 (URN)10.1074/mcp.M112.026757 (DOI)000330536400021 ()2-s2.0-84884389479 (Scopus ID)
Funder
VinnovaKnut and Alice Wallenberg FoundationAFA InsuranceSwedish Research CouncilScience for Life Laboratory - a national resource center for high-throughput molecular bioscience
Note

QC 20131204

Available from: 2013-12-04 Created: 2013-11-11 Last updated: 2017-12-06Bibliographically approved
5. Autoantibody targets in vaccine-associated narcolepsy
Open this publication in new window or tab >>Autoantibody targets in vaccine-associated narcolepsy
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(English)Manuscript (preprint) (Other academic)
National Category
Biomedical Laboratory Science/Technology
Identifiers
urn:nbn:se:kth:diva-158942 (URN)
Note

QS 2015

Available from: 2015-01-15 Created: 2015-01-15 Last updated: 2015-01-16Bibliographically approved

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Häggmark, Anna

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