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Affinity Proteomic Profiling of Plasma, Cerebrospinal Fluid, and Brain Tissue within Multiple Sclerosis
KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.ORCID-id: 0000-0001-7843-2960
KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.ORCID-id: 0000-0002-0056-1313
KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.ORCID-id: 0000-0001-8603-8293
Visa övriga samt affilieringar
Antal upphovsmän: 162014 (Engelska)Ingår i: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 13, nr 11, s. 4607-4619Artikel i tidskrift (Refereegranskat) 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.

Ort, förlag, år, upplaga, sidor
American Chemical Society (ACS), 2014. Vol. 13, nr 11, s. 4607-4619
Nyckelord [en]
antibodies, suspension bead arrays, plasma, CSF, brain tissue, immunofluorescence, multiple sclerosis
Nationell ämneskategori
Biokemi och molekylärbiologi
Identifikatorer
URN: urn:nbn:se:kth:diva-150383DOI: 10.1021/pr500609eISI: 000344636500012PubMedID: 25231264Scopus ID: 2-s2.0-84908890596OAI: oai:DiVA.org:kth-150383DiVA, id: diva2:742773
Forskningsfinansiär
VetenskapsrådetAFA FörsäkringScience for Life Laboratory - a national resource center for high-throughput molecular bioscienceKnut och Alice Wallenbergs Stiftelse
Anmärkning

QC 20141212

Updated from manuscript to article in journal.

Tillgänglig från: 2014-09-02 Skapad: 2014-09-02 Senast uppdaterad: 2017-12-05Bibliografiskt granskad
Ingår i avhandling
1. Affinity Arrays for Profiling Proteins and Autoantibody Repertoires
Öppna denna publikation i ny flik eller fönster >>Affinity Arrays for Profiling Proteins and Autoantibody Repertoires
2014 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
Ort, förlag, år, upplaga, sidor
Stockholm: KTH Royal Institute of Technology, 2014. s. xi, 159
Serie
TRITA-BIO-Report, ISSN 1654-2312 ; 2014:12
Nationell ämneskategori
Biomedicinsk laboratorievetenskap/teknologi
Identifikatorer
urn:nbn:se:kth:diva-150398 (URN)978-91-7595-209-3 (ISBN)
Disputation
2014-09-05, Inghesalen, Karolinska Institute, Tomtebodavägen 18 A, Solna, 10:00 (Engelska)
Opponent
Handledare
Forskningsfinansiär
Science for Life Laboratory - a national resource center for high-throughput molecular bioscience
Anmärkning

QC 20140902

Tillgänglig från: 2014-09-02 Skapad: 2014-09-02 Senast uppdaterad: 2014-09-02Bibliografiskt granskad
2. Neuroproteomic profiling of human body fluids
Öppna denna publikation i ny flik eller fönster >>Neuroproteomic profiling of human body fluids
2015 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
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.

Ort, förlag, år, upplaga, sidor
Stockholm: KTH Royal Institute of Technology, 2015. s. viii, 67
Serie
TRITA-BIO-Report, ISSN 1654-2312 ; 2015:2
Nationell ämneskategori
Medicinsk bioteknologi (med inriktning mot cellbiologi (inklusive stamcellsbiologi), molekylärbiologi, mikrobiologi, biokemi eller biofarmaci)
Forskningsämne
Bioteknologi
Identifikatorer
urn:nbn:se:kth:diva-158944 (URN)978-91-7595-402-8 (ISBN)
Disputation
2015-02-06, Rockefellersalen, KI, Solna, 09:00 (Engelska)
Opponent
Handledare
Anmärkning

QC 20150116

Tillgänglig från: 2015-01-16 Skapad: 2015-01-15 Senast uppdaterad: 2015-01-16Bibliografiskt granskad
3. Affinity assays for profiling disease-associated proteins in human plasma
Öppna denna publikation i ny flik eller fönster >>Affinity assays for profiling disease-associated proteins in human plasma
2017 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
Abstract [en]

Affinity-based proteomics offers opportunities for the discovery and validation of disease-associated proteins in human body fluids. This thesis describes the use of antibody-based immunoassays for multiplexed analysis of proteins in human plasma, serum and cerebrospinal fluid (CSF). This high-throughput method was applied with the objective to identify proteins associated to clinical variables. The main work in this thesis was conducted within the diseases of multiple sclerosis and malignant melanoma, as well as mammographic density, a risk factor for breast cancer.

The suspension bead array (SBA) technology has been the main method for the work presented in this thesis (Paper I-IV). SBA assays and other affinity proteomic technologies were introduced for protein profiling of sample material obtained from clinical collaborators and biobanks. Perspectives on the validation of antibody selectivity by means of e.g. immuno-capture mass spectrometry are also provided.

Paper I describes the development and application of a protocol for multiplexed pro- tein profiling of CSF. The analysis of 340 CSF samples from patients with multiple sclerosis and other neurological disease revealed proteins with potential association to disease progression (GAP43) and inflammation (SERPINA3). Paper II continued on this work with an extended investigation of more than 1,000 clinical samples and included both plasma and CSF collected from the same patients. Comparison of disease subtypes and controls revealed five plasma proteins of potential diagnostic relevance, such as IRF8 and GAP43. The previously reported associations for GAP43 and SERPINA3 in CSF was confirmed. Subsequent immunohistochemical analysis of post-mortem brain tissue revealed differential protein expression in disease affected areas. In Paper III, 150 serum samples from patients with cutaneous malignant melanoma were analyzed. Protein profiles from antibody bead arrays suggested three proteins (RGN, MTHFD1L, STX7) of differential abundance between patients with no disease recurrence and low tumor thickness (T-stage 1 and 2) compared to patients with high tumor thickness (T-stage 3 and 4) and disease recurrence. We observed MTHFD1L expression in tissue of a majority of patients, while expression of STX7 in melanoma tissue had been reported previously. Paper IV describes the analysis of protein in plasma in relation to mammographic breast density (MD), one of the strongest risk factors for the development of breast cancers. More than 1,300 women without prior history of breast cancer were screened. Linear associations to MD in two independent sample sets were found for 11 proteins, which are expressed in the breast and involved in tissue homeostasis, DNA repair, cancer development and/or progression in MD.

In conclusion, this thesis describes the use of multiplexed antibody bead arrays for protein profiling of serum, plasma and CSF, and it shortlists disease associated proteins for further validation studies. 

Ort, förlag, år, upplaga, sidor
Stockholm: KTH Royal Institute of Technology, 2017. s. 89
Serie
TRITA-BIO-Report, ISSN 1654-2312 ; 2017:6
Nyckelord
proteomics, affinity proteomics, immunoassay, antibody microarray, suspension bead array, protein profiling, immuno-capture, plasma, serum, cerebrospinal fluid, biomarker discovery, multiple sclerosis, malignant melanoma, mammographic breast density
Nationell ämneskategori
Medicinsk bioteknologi (med inriktning mot cellbiologi (inklusive stamcellsbiologi), molekylärbiologi, mikrobiologi, biokemi eller biofarmaci)
Forskningsämne
Bioteknologi
Identifikatorer
urn:nbn:se:kth:diva-202616 (URN)978-91-7729-297-5 (ISBN)
Disputation
2017-03-31, Inghesalen, KI, Tomtebodavägen 18A, Solna, 10:00 (Engelska)
Opponent
Handledare
Anmärkning

QC 20170302

Tillgänglig från: 2017-03-02 Skapad: 2017-03-02 Senast uppdaterad: 2017-03-06Bibliografiskt granskad

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Byström, SannaAyoglu, BurcuHäggmark, AnnaHong, Mun-GwanDrobin, KimForsström, BjörnFredolini, ClaudiaUhlén, MathiasNilsson, PeterSchwenk, Jochen M.
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