| 1. |
- Darmanis, Spyros, et al.
(författare)
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ProteinSeq : high-performance proteomic analyses by proximity ligation and next generation sequencing
- 2011
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 6:9, s. e25583-
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Tidskriftsartikel (refereegranskat)abstract
- Despite intense interest, methods that provide enhanced sensitivity and specificity in parallel measurements of candidate protein biomarkers in numerous samples have been lacking. We present herein a multiplex proximity ligation assay with readout via realtime PCR or DNA sequencing (ProteinSeq). We demonstrate improved sensitivity over conventional sandwich assays for simultaneous analysis of sets of 35 proteins in 5 μl of blood plasma. Importantly, we observe a minimal tendency to increased background with multiplexing, compared to a sandwich assay, suggesting that higher levels of multiplexing are possible. We used ProteinSeq to analyze proteins in plasma samples from cardiovascular disease (CVD) patient cohorts and matched controls. Three proteins, namely P-selectin, Cystatin-B and Kallikrein-6, were identified as putative diagnostic biomarkers for CVD. The latter two have not been previously reported in the literature and their potential roles must be validated in larger patient cohorts. We conclude that ProteinSeq is promising for screening large numbers of proteins and samples while the technology can provide a much-needed platform for validation of diagnostic markers in biobank samples and in clinical use.
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| 2. |
- Darmanis, Spyros, et al.
(författare)
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Multiplexed solid-phase proximity ligation assays: Highly specific and parallel protein measurements with DNA sequencing readout
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Identification and validation of protein biomarkers is a very important step towards the understanding of the underlying mechanisms of disease, early diagnosis and efficient patient treatment. To carry out this task, methods are needed that would allow us to mine the proteome with sufficient sensitivity and specificity in large sets of samples. We present herein the development of a Multiplexed Proximity Ligation Assay (MultiPLAy), to facilitate efficient protein profiling in a parallel, sensitive and specific manner. We showed that for the simultaneous analysis of 35 proteins MultiPLAy exhibited an improved sensitivity over conventional sandwich assays as well as a smaller susceptibility to background signal increase in the transition from singleplex to multiplex. We used MultiPLAy to identify putative biomarkers in two separate sample cohorts of colorectal cancer (CRC) and cardiovascular disease (CVD) and with the use a novel multivariate analysis approach were able to identify new, as well as already known diagnostic biomarkers. Furthermore we were able to combine MultiPLAy with the use of next-generation sequencing allowing for the first time digital recording of protein profiles in blood. We demonstrated good reproducibility of MultiPLAy coupled to next-generation sequencing, as well as a satisfactory correlation to standard real-time PCR readout. We conclude that MultiPLAy has great potential as a basis for highly multiplexed protein detection assays that can be utilized for the identification of large numbers of proteins or protein variants. This will allow extensive validation of protein expression patterns in biobanked samples and in prospective studies, and can provide a much-needed platform for efficient validation of diagnostic markers for clinical use.
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| 3. |
- Darmanis, Spyros, et al.
(författare)
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Simultaneous Multiplexed Measurement of RNA and Proteins in Single Cells
- 2016
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Ingår i: Cell Reports. - : Elsevier BV. - 2211-1247. ; 14:2, s. 380-389
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Tidskriftsartikel (refereegranskat)abstract
- Significant advances have been made in methods to analyze genomes and transcriptomes of single cells, but to fully define cell states, proteins must also be accessed as central actors defining a cell's phenotype. Methods currently used to analyze endogenous protein expression in single cells are limited in specificity, throughput, or multiplex capability. Here, we present an approach to simultaneously and specifically interrogate large sets of protein and RNA targets in lysates from individual cells, enabling investigations of cell functions and responses. We applied our method to investigate the effects of BMP4, an experimental therapeutic agent, on early-passage glioblastoma cell cultures. We uncovered significant heterogeneity in responses to treatment at levels of RNA and protein, with a subset of cells reacting in a distinct manner to BMP4. Moreover, we found overall poor correlation between protein and RNA at the level of single cells, with proteins more accurately defining responses to treatment.
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| 4. |
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| 5. |
- Fredriksson, Simon, et al.
(författare)
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Protein detection using proximity-dependent DNA ligation assays
- 2002
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Ingår i: Nature Biotechnology. - : Springer Science and Business Media LLC. - 1087-0156 .- 1546-1696. ; 20:5, s. 473-477
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Tidskriftsartikel (refereegranskat)abstract
- The advent of in vitro DNA amplification has enabled rapid acquisition of genomic information. We present here an analogous technique for protein detection, in which the coordinated and proximal binding of a target protein by two DNA aptamers promotes ligation of oligonucleotides linked to each aptamer affinity probe. The ligation of two such proximity probes gives rise to an amplifiable DNA sequence that reflects the identity and amount of the target protein. This proximity ligation assay detects zeptomole (40 x 10(-21) mol) amounts of the cytokine platelet-derived growth factor (PDGF) without washes or separations, and the mechanism can be generalized to other forms of protein analysis.
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| 6. |
- Genshaft, Alex S., et al.
(författare)
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Multiplexed, targeted profiling of single-cell proteomes and transcriptomes in a single reaction
- 2016
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Ingår i: Genome Biology. - : Springer Science and Business Media LLC. - 1465-6906 .- 1474-760X. ; 17
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Tidskriftsartikel (refereegranskat)abstract
- We present a scalable, integrated strategy for coupled protein and RNA detection from single cells. Our approach leverages the DNA polymerase activity of reverse transcriptase to simultaneously perform proximity extension assays and complementary DNA synthesis in the same reaction. Using the Fluidigm C1 (TM) system, we profile the transcriptomic and proteomic response of a human breast adenocarcinoma cell line to a chemical perturbation, benchmarking against in situ hybridizations and immunofluorescence staining, as well as recombinant proteins, ERCC Spike-Ins, and population lysate dilutions. Through supervised and unsupervised analyses, we demonstrate synergies enabled by simultaneous measurement of single-cell protein and RNA abundances. Collectively, our generalizable approach highlights the potential for molecular metadata to inform highly-multiplexed single-cell analyses.
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| 7. |
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| 8. |
- Gullberg, Mats, et al.
(författare)
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Cytokine detection by antibody-based proximity ligation
- 2004
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 101:22, s. 8420-4
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- Efficient and precise detection techniques, along with extensive repertoires of specific binding reagents, will be needed to meet the challenges of proteome analyses. The recently established proximity ligation mechanism enables sensitive high-capacity protein measurements by converting the detection of specific proteins to the analysis of DNA sequences. Proximity probes containing oligonucleotide extensions are designed to bind pairwise to target proteins and to form amplifiable tag sequences by ligation when brought in proximity. In our previous report, both the ligatable arms and the protein binders were DNA molecules. We now generalize the method by providing simple and convenient protocols to convert any polyclonal antibodies or matched pair of monoclonal antibodies to proximity probe sets through the attachment of oligonucleotide sequences. Sufficient reagent for >100,000 proximity ligation assays can be prepared from 1 microg of antibody. The technique is applied to measure cytokines in a homogenous test format with femtomolar detection sensitivities in 1-microl samples, and we exemplify its utility in situations when only minute sample amounts are available.
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| 9. |
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| 10. |
- Gustafsdottir, Sigrun M., et al.
(författare)
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Detection of individual microbial pathogens by proximity ligation
- 2006
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Ingår i: Clinical Chemistry. - : Oxford University Press (OUP). - 0009-9147 .- 1530-8561. ; 52:6, s. 1152-1160
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: Nucleic acid amplification allows the detection of single infectious agents. Protein-based assays, although they provide information on ongoing infections, have substantially less detection sensitivity.METHODS: We used proximity ligation reactions to detect proteins on bacteria and virus particles via nucleic acid amplification. Antibodies recognizing viral or bacterial surface proteins were equipped with DNA strands that could be joined by ligation when several antibodies were bound in proximity to surface proteins of individual infectious agents.RESULTS: Detection sensitivities similar to those of nucleic acid-based detection reactions were achieved directly in infected samples for a parvovirus and an intracellular bacterium.CONCLUSIONS: This method enables detection of ligated DNA strands with good sensitivity by real-time PCR and could be of value for early diagnosis of infectious disease and in biodefense.
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