| 1. |
- Gloriam, David E., et al.
(författare)
-
A Community Standard Format for the Representation of Protein Affinity Reagents
- 2010
-
Ingår i: Molecular & Cellular Proteomics. - 1535-9476 .- 1535-9484. ; 9:1, s. 1-10
-
Tidskriftsartikel (refereegranskat)abstract
- Protein affinity reagents (PARs), most commonly antibodies, are essential reagents for protein characterization in basic research, biotechnology, and diagnostics as well as the fastest growing class of therapeutics. Large numbers of PARs are available commercially; however, their quality is often uncertain. In addition, currently available PARs cover only a fraction of the human proteome, and their cost is prohibitive for proteome scale applications. This situation has triggered several initiatives involving large scale generation and validation of antibodies, for example the Swedish Human Protein Atlas and the German Antibody Factory. Antibodies targeting specific subproteomes are being pursued by members of Human Proteome Organisation (plasma and liver proteome projects) and the United States National Cancer Institute (cancer-associated antigens). ProteomeBinders, a European consortium, aims to set up a resource of consistently quality-controlled protein-binding reagents for the whole human proteome. An ultimate PAR database resource would allow consumers to visit one online warehouse and find all available affinity reagents from different providers together with documentation that facilitates easy comparison of their cost and quality. However, in contrast to, for example, nucleotide databases among which data are synchronized between the major data providers, current PAR producers, quality control centers, and commercial companies all use incompatible formats, hindering data exchange. Here we propose Proteomics Standards Initiative (PSI)-PAR as a global community standard format for the representation and exchange of protein affinity reagent data. The PSI-PAR format is maintained by the Human Proteome Organisation PSI and was developed within the context of ProteomeBinders by building on a mature proteomics standard format, PSI-molecular interaction, which is a widely accepted and established community standard for molecular interaction data. Further information and documentation are available on the PSI-PAR web site. Molecular & Cellular Proteomics 9: 1-10, 2010.
|
|
| 2. |
- Grannas, Karin, 1983-
(författare)
-
Improvements and Applications of in situ Proximity Ligation Assays
- 2015
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The cells building up the human body is in constant communication with each other. This communication is done through large complex networks of signaling pathways for inter- and intracellular signal transduction. The signaling activity regulates many important processes, for example cell death, proliferation and differentiation. Information within the signaling networks is communicated over the cell membrane, through the cytoplasm and entering the nucleus by protein activities such as protein-protein interactions (PPIs) and post translation modifications (PTMs). The cells adapts to their own environment, responding to multiple stimuli from their surroundings. This in combination with memory of previous responses, difference in cell cycles stages and sometimes altered genetic background generates heterogeneous cell populations in which every cell is slightly different from its neighbor. This calls for methods to study the activity of endogenous proteins in individual cells within a population.In situ proximity ligation assay (in situ PLA) was originally developed to visualize interaction between endogenous proteins in fixed cells and tissue and can also be applied to detect PTMs. This thesis describe the application of in situ PLA to study PPIs in signaling pathways and the work to further develop and improve techniques for proximity dependent detection. In paper I in situ PLA is used to study cross talk between the Hippo and the TGFβ signaling pathways. The study shows the complex formation by the transcription co-factors of the Hippo pathway, Yap and Taz, and the main effectors of the TGFβ pathway Smad2/3. Furthermore the density dependent localization of the interaction is described.Paper II presents a new version of the in situ PLA probes for simultaneous detection of multiple complexes. Visualization of various complexes involving EGFR, Her2 and Her3 is presented as a proof of concept.The efficiency of in situ PLA is limited by several factors, one being the design of PLA probes and oligonucleotide systems. Even upon proximal binding of the probes there is a risk of formation of non-circular ligation products, which cannot be amplified and detected. In Paper III two new PLA probes are presented aiming to reduce the formation of non-circular ligation product and hence increase the detection efficiency of in situ PLA.Paper IV presents a new method for detection of protein complexes and phosphorylation; proxHCR. ProxHCR combines signal amplification by enzyme free hybridization chain reaction (HCR) with the requirement of proximal binding of two affinity probes. As a proof of principle the method is used to detect multiple complexes and protein phosphorylation in fixed cells and tissue.
|
|
| 3. |
- Brofelth, Mattias, et al.
(författare)
-
Multiplex profiling of serum proteins in solution using barcoded antibody fragments and next generation sequencing
- 2020
-
Ingår i: Communications Biology. - : NATURE PUBLISHING GROUP. - 2399-3642. ; 3:1
-
Tidskriftsartikel (refereegranskat)abstract
- The composition of serum proteins is reflecting the current health status and can, with the right tools, be used to detect early signs of disease, such as an emerging cancer. An earlier diagnosis of cancer would greatly increase the chance of an improved outcome for the patients. However, there is still an unmet need for proficient tools to decipher the information in the blood proteome, which calls for further technological development. Here, we present a proof-of-concept study that demonstrates an alternative approach for multiplexed protein profiling of serum samples in solution, using DNA barcoded scFv antibody fragments and next generation sequencing. The outcome shows high accuracy when discriminating samples derived from pancreatic cancer patients and healthy controls and represents a scalable alternative for serum analysis. Brofelth, Ekstrand et al use DNA barcoded scFv antibody fragments and next generation sequencing for multiplex profiling of proteins in serum from pancreatic cancer patients with high accuracy. This approach can potentially be used in high throughput precision diagnosis.
|
|
| 4. |
- Gunnarsson, Anders, 1981, et al.
(författare)
-
Kinetics of Ligand Binding to Membrane Receptors from Equilibrium Fluctuation Analysis of Single Binding Events
- 2011
-
Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 1520-5126 .- 0002-7863. ; 133:38, s. 14852-14855
-
Tidskriftsartikel (refereegranskat)abstract
- Equilibrium fluctuation analysis of single binding events has been used to extract binding kinetics of ligand interactions with cell-membrane bound receptors. Time-dependent total internal reflection fluorescence (TIRF) imaging was used to extract residence-time statistics of fluorescently stained liposomes derived directly from cell membranes upon their binding to surface-immobilized antibody fragments. The dissociation rate constants for two pharmaceutical relevant antibodies directed against different B-cell expressed membrane proteins was clearly discriminated, and the affinity of the interaction could be determined by inhibiting the interaction with increasing concentrations of soluble antibodies. The single-molecule sensitivity made the analysis possible without overexpressed membrane proteins, which makes the assay attractive in early drug-screening applications.
|
|
| 5. |
- Brofelth, Mattias, et al.
(författare)
-
Site-specific photocoupling of pBpa mutated scFv antibodies for use in affinity proteomics
- 2017
-
Ingår i: Biochimica et Biophysica Acta - Proteins and Proteomics. - : Elsevier BV. - 1570-9639. ; 1865:8, s. 985-996
-
Tidskriftsartikel (refereegranskat)abstract
- Recombinant antibody libraries can provide a source of renewable and high-performing binders tailored for use in affinity proteomics. In this context, the process of generating site-specific 1:1 tagging/functionalization and/or orientated surface immobilization of antibodies has, however, proved to be challenging. Hence, novel ways of generating such engineered antibodies for use in affinity proteomics could have a major impact on array performance. In this study, we have further tailored the design of human recombinant scFv antibodies for site-specific photocoupling through the use of an unnatural amino acid (UAA) and the Dock'n'Flash technology. In more detail, we have generated the 2nd generation of scFvs carrying the photoreactive UAA p-benzoyl-l-phenylalanine (pBpa). Based on key properties, such as expression levels, activity, and affinity, a preferred choice of site for pBpa, located in the beginning of the C-terminal affinity-tag, was for the first time pin-pointed. Further, the results showed that pBpa mutated antibody could be site-specifically photocoupled to free and surface immobilized β-cyclodextrin (an affinity ligand to pBpa). This paves the way for use of scFv antibodies, engineered for site-specific photochemical-based tagging, functionalization, and orientated surface immobilization, in affinity proteomics.
|
|
| 6. |
- Ingvarsson, Johan, et al.
(författare)
-
Detection of pancreatic cancer using antibody microarray-based serum protein profiling
- 2008
-
Ingår i: Proteomics. - : Wiley. - 1615-9861 .- 1615-9853. ; 8:11, s. 2211-2219
-
Tidskriftsartikel (refereegranskat)abstract
- The driving force behind oncoproteomics is to identify protein signatures that are associated with a particular malignancy. Here, we have used a recombinant scFv antibody microarray in an attempt to classify sera derived from pancreatic adenocarcinoma patients versus healthy subjects. Based on analysis of nonfractionated, directly labeled, whole human serum proteomes we have identified a protein signature based on 19 nonredundant analytes, that discriminates between cancer patients and healthy subjects. Furthermore, a potential protein signature, consisting of 21 protein analytes, could be defined that was shown to be associated with cancer patients having a life expectancy of <12 months. Taken together, the data suggest that antibody microarray analysis of complex proteomes will be a useful tool to define disease associated protein signatures.
|
|
| 7. |
|
|
| 8. |
- Steinhauer, Cornelia, et al.
(författare)
-
Biocompatability of surfaces for antibody microarrays: Design of macroporous silicon substrates
- 2005
-
Ingår i: Micro Total Analysis Systems 2004, Vol 2. - 0260-6291. ; :297, s. 121-123
-
Konferensbidrag (refereegranskat)abstract
- Antibody microarray is a novel technology with great promise within proteomics. Intense work is under way to evolve this methodology into the high-throughput proteomic research tool needed by the research community. Despite recent advances, there is a growing need for additional highperformance substrates for antibody microarrays as well as for protein arrays in general. In this study, we have sucessfully designed novel, highly biocompatible and well-performing silicon-based supports that has the capacity to play a significant role within current and future antibody and protein microarray applications within the field of proteomics.
|
|
| 9. |
|
|
| 10. |
- Ellmark, Peter, et al.
(författare)
-
Attovial-based antibody nanoarrays.
- 2009
-
Ingår i: Proteomics. - : Wiley. - 1615-9861 .- 1615-9853. ; 9:24, s. 5406-5413
-
Tidskriftsartikel (refereegranskat)abstract
- Antibody array-based technology is a powerful emerging tool in proteomics, but to enable global proteome analysis, antibody array layouts with even higher density has to be developed. To this end, we have further developed the first generation of a nanoarray platform, based on attoliter sized vials, attovials, which we have characterized and used for the detection of complement factor C1q in human serum samples. Finally, we demonstrated proof-of-concept for individual functionalisation of the attovials with a recombinant antibody.
|
|
