| 1. |
- Darmanis, Spyros, et al.
(författare)
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Identification of Candidate Serum Proteins for Classifying Well-Differentiated Small Intestinal Neuroendocrine Tumors
- 2013
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 8:11, s. e81712-
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Tidskriftsartikel (refereegranskat)abstract
- BackgroundPatients with well-differentiated small intestine neuroendocrine tumors (WD-SI-NET) are most often diagnosed at a metastatic stage of disease, which reduces possibilities for a curative treatment. Thus new approaches for earlier detection and improved monitoring of the disease are required.Materials and methodsSuspension bead arrays targeting 124 unique proteins with antibodies from the Human Protein Atlas were used to profile biotinylated serum samples. Discoveries from a cohort of 77 individuals were followed up in a cohort of 132 individuals both including healthy controls as well as patients with untreated primary WD-SI-NETs, lymph node metastases and liver metastases.Results A set of 20 antibodies suggested promising proteins for further verification based on technically verified statistical significance. Proceeding, we assessed the classification performance in an independent cohort of patient serum, achieving, classification accuracy of up to 85% with different subsets of antibodies in respective pairwise group comparisons. The protein profiles of nine targets, namely IGFBP2, IGF1, SHKBP1, ETS1, IL1α, STX2, MAML3, EGR3 and XIAP were verified as significant contributors to tumor classification.ConclusionsWe propose new potential protein biomarker candidates for classifying WD-SI-NET at different stage of disease. Further evaluation of these proteins in larger sample sets and with alternative approaches is needed in order to further improve our understanding of their functional relation to WD-SI-NET and their eventual use in diagnostics.
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| 2. |
- Uhlén, Mathias, et al.
(författare)
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The human secretome
- 2019
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Ingår i: Science Signaling. - : American Association for the Advancement of Science (AAAS). - 1945-0877 .- 1937-9145. ; 12:609
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Tidskriftsartikel (refereegranskat)abstract
- The proteins secreted by human cells (collectively referred to as the secretome) are important not only for the basic understanding of human biology but also for the identification of potential targets for future diagnostics and therapies. Here, we present a comprehensive analysis of proteins predicted to be secreted in human cells, which provides information about their final localization in the human body, including the proteins actively secreted to peripheral blood. The analysis suggests that a large number of the proteins of the secretome are not secreted out of the cell, but instead are retained intracellularly, whereas another large group of proteins were identified that are predicted to be retained locally at the tissue of expression and not secreted into the blood. Proteins detected in the human blood by mass spectrometry-based proteomics and antibody-based immuno-assays are also presented with estimates of their concentrations in the blood. The results are presented in an updated version 19 of the Human Protein Atlas in which each gene encoding a secretome protein is annotated to provide an open-access knowledge resource of the human secretome, including body-wide expression data, spatial localization data down to the single-cell and subcellular levels, and data about the presence of proteins that are detectable in the blood.
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| 3. |
- Fagerberg, Linn, et al.
(författare)
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Analysis of the human tissue-specific expression by genome-wide integration of transcriptomics and antibody-based proteomics
- 2014
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Ingår i: Molecular & Cellular Proteomics. - 1535-9476 .- 1535-9484. ; 13:2, s. 397-406
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Tidskriftsartikel (refereegranskat)abstract
- Global classification of the human proteins with regards to spatial expression patterns across organs and tissues is important for studies of human biology and disease. Here, we used a quantitative transcriptomics analysis (RNA-Seq) to classify the tissue-specific expression of genes across a representative set of all major human organs and tissues and combined this analysis with antibody- based profiling of the same tissues. To present the data, we launch a new version of the Human Protein Atlas that integrates RNA and protein expression data corresponding to 80% of the human protein-coding genes with access to the primary data for both the RNA and the protein analysis on an individual gene level. We present a classification of all human protein-coding genes with regards to tissue-specificity and spatial expression pattern. The integrative human expression map can be used as a starting point to explore the molecular constituents of the human body.
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| 4. |
- Abdellah, Tebani, et al.
(författare)
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Integration of molecular profiles in a longitudinal wellness profiling cohort.
- 2020
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Ingår i: Nature communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 11:1
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Tidskriftsartikel (refereegranskat)abstract
- An important aspect of precision medicine is to probe the stability in molecular profiles among healthy individuals over time. Here, we sample a longitudinal wellness cohort with 100 healthy individuals and analyze blood molecular profiles including proteomics, transcriptomics, lipidomics, metabolomics, autoantibodies andimmune cell profiling, complementedwith gut microbiota composition and routine clinical chemistry. Overall, our results show high variation between individuals across different molecular readouts, while the intra-individual baseline variation is low. The analyses show that each individual has a unique and stable plasma protein profile throughout the study period and that many individuals also show distinct profiles with regards to the other omics datasets, with strong underlying connections between the blood proteome and the clinical chemistry parameters. In conclusion, the results support an individual-based definition of health and show that comprehensive omics profiling in a longitudinal manner is a path forward for precision medicine.
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| 5. |
- Uhlén, Mathias, et al.
(författare)
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Tissue-based map of the human proteome
- 2015
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Ingår i: Science. - : American Association for the Advancement of Science (AAAS). - 0036-8075 .- 1095-9203. ; 347:6220, s. 1260419-
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Tidskriftsartikel (refereegranskat)abstract
- Resolving the molecular details of proteome variation in the different tissues and organs of the human body will greatly increase our knowledge of human biology and disease. Here, we present a map of the human tissue proteome based on an integrated omics approach that involves quantitative transcriptomics at the tissue and organ level, combined with tissue microarray-based immunohistochemistry, to achieve spatial localization of proteins down to the single-cell level. Our tissue-based analysis detected more than 90% of the putative protein-coding genes. We used this approach to explore the human secretome, the membrane proteome, the druggable proteome, the cancer proteome, and the metabolic functions in 32 different tissues and organs. All the data are integrated in an interactive Web-based database that allows exploration of individual proteins, as well as navigation of global expression patterns, in all major tissues and organs in the human body.
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| 6. |
- Fredolini, Claudia, et al.
(författare)
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Systematic assessment of antibody selectivity in plasma based on a resource of enrichment profiles
- 2019
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Ingår i: Scientific Reports. - : Nature Publishing Group. - 2045-2322. ; 9
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Tidskriftsartikel (refereegranskat)abstract
- There is a strong need for procedures that enable context and application dependent validation of antibodies. Here, we applied a magnetic bead assisted workflow and immunoprecipitation mass spectrometry (IP-MS/MS) to assess antibody selectivity for the detection of proteins in human plasma. A resource was built on 414 IP experiments using 157 antibodies (targeting 120 unique proteins) in assays with heat-treated or untreated EDTA plasma. For each protein we determined their antibody related degrees of enrichment using z-scores and their frequencies of identification across all IP assays. Out of 1,313 unique endogenous proteins, 426 proteins (33%) were detected in >20% of IPs, and these background components were mainly comprised of proteins from the complement system. For 45% (70/157) of the tested antibodies, the expected target proteins were enriched (z-score >= 3). Among these 70 antibodies, 59 (84%) co-enriched other proteins beside the intended target and mainly due to sequence homology or protein abundance. We also detected protein interactions in plasma, and for IGFBP2 confirmed these using several antibodies and sandwich immunoassays. The protein enrichment data with plasma provide a very useful and yet lacking resource for the assessment of antibody selectivity. Our insights will contribute to a more informed use of affinity reagents for plasma proteomics assays.
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| 7. |
- Byström, Sanna
(författare)
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Affinity assays for profiling disease-associated proteins in human plasma
- 2017
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- 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.
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| 8. |
- Häggmark, Anna, 1985-
(författare)
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Neuroproteomic profiling of human body fluids
- 2015
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- 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.
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| 9. |
- Neiman, Maja, et al.
(författare)
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Selectivity analysis of single binder assays used in plasma protein profiling
- 2013
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Ingår i: Proteomics. - : Wiley. - 1615-9853 .- 1615-9861. ; 13:23-24, s. 3406-3410
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Tidskriftsartikel (refereegranskat)abstract
- The increasing availability of antibodies toward human proteins enables broad explorations of the proteomic landscape in cells, tissues, and body fluids. This includes assays with antibody suspension bead arrays that generate protein profiles of plasma samples by flow cytometer analysis. However, antibody selectivity is context dependent so it is necessary to corroborate on-target detection over off-target binding. To address this, we describe a concept to directly verify interactions from antibody-coupled beads by analysis of their eluates by Western blots and MS. We demonstrate selective antibody binding in complex samples with antibodies toward a set of chosen proteins with different abundance in plasma and serum, and illustrate the need to adjust sample and bead concentrations accordingly. The presented approach will serve as an important tool for resolving differential protein profiles from antibody arrays within plasma biomarker discoveries.
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| 10. |
- Dahl, Leo, 1995-, et al.
(författare)
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Multiplexed selectivity screening of anti-GPCR antibodies
- 2023
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Ingår i: Science Advances. - : American Association for the Advancement of Science (AAAS). - 2375-2548. ; 9:18
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Tidskriftsartikel (refereegranskat)abstract
- G protein-coupled receptors (GPCRs) control critical cellular signaling pathways. Therapeutic agents including anti-GPCR antibodies (Abs) are being developed to modulate GPCR function. However, validating the selectivity of anti-GPCR Abs is challenging because of sequence similarities among individual receptors within GPCR sub-families. To address this challenge, we developed a multiplexed immunoassay to test >400 anti-GPCR Abs from the Human Protein Atlas targeting a customized library of 215 expressed and solubilized GPCRs representing all GPCR subfamilies. We found that-61% of Abs tested were selective for their intended target,-11% bound off -target, and-28% did not bind to any GPCR. Antigens of on-target Abs were, on average, significantly longer, more disordered, and less likely to be buried in the interior of the GPCR protein than the other Abs. These results provide important insights into the immunogenicity of GPCR epitopes and form a basis for designing therapeu-tic Abs and for detecting pathological auto-Abs against GPCRs.
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