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Affinity assays for profiling disease-associated proteins in human plasma
KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
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. 

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2017. , p. 89
Series
TRITA-BIO-Report, ISSN 1654-2312 ; 2017:6
Keywords [en]
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
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-202616ISBN: 978-91-7729-297-5 (print)OAI: oai:DiVA.org:kth-202616DiVA, id: diva2:1078049
Public defence
2017-03-31, Inghesalen, KI, Tomtebodavägen 18A, Solna, 10:00 (English)
Opponent
Supervisors
Note

QC 20170302

Available from: 2017-03-02 Created: 2017-03-02 Last updated: 2017-03-06Bibliographically 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, p. 2256-2267Article 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.

Keywords
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, p. 4607-4619Article 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
Keywords
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. Affinity proteomics exploration of melanoma identifies proteins in serum with associations to T-stage and recurrence
Open this publication in new window or tab >>Affinity proteomics exploration of melanoma identifies proteins in serum with associations to T-stage and recurrence
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(English)Manuscript (preprint) (Other academic)
National Category
Cancer and Oncology Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:kth:diva-202614 (URN)
Note

QC 20170302

Available from: 2017-03-02 Created: 2017-03-02 Last updated: 2017-03-13Bibliographically approved
4. Affinity proteomic profiling of plasma for proteins associated to mammographic breast density
Open this publication in new window or tab >>Affinity proteomic profiling of plasma for proteins associated to mammographic breast density
Show others...
(English)Manuscript (preprint) (Other academic)
National Category
Cancer and Oncology Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:kth:diva-202615 (URN)
Note

QC 20170302

Available from: 2017-03-02 Created: 2017-03-02 Last updated: 2017-03-13Bibliographically approved

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