| 1. |
- Azimi, Alireza, et al.
(författare)
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Targeting CDK2 overcomes melanoma resistance against BRAF and Hsp90 inhibitors
- 2018
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Ingår i: Molecular Systems Biology. - : EMBO. - 1744-4292. ; 14:3
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Tidskriftsartikel (refereegranskat)abstract
- Novel therapies are undergoing clinical trials, for example, the Hsp90 inhibitor, XL888, in combination with BRAF inhibitors for the treatment of therapy-resistant melanomas. Unfortunately, our data show that this combination elicits a heterogeneous response in a panel of melanoma cell lines including PDX-derived models. We sought to understand the mechanisms underlying the differential responses and suggest a patient stratification strategy. Thermal proteome profiling (TPP) identified the protein targets of XL888 in a pair of sensitive and unresponsive cell lines. Unbiased proteomics and phosphoproteomics analyses identified CDK2 as a driver of resistance to both BRAF and Hsp90 inhibitors and its expression is regulated by the transcription factor MITF upon XL888 treatment. The CDK2 inhibitor, dinaciclib, attenuated resistance to both classes of inhibitors and combinations thereof. Notably, we found that MITF expression correlates with CDK2 upregulation in patients; thus, dinaciclib would warrant consideration for treatment of patients unresponsive to BRAF-MEK and/or Hsp90 inhibitors and/or harboring MITF amplification/overexpression.
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| 2. |
- Edfors, Fredrik, 1988-, et al.
(författare)
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A recombinant protein standard resource for targeted proteomics
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Here, we have used a resource of 26,000 recombinant protein fragments to create custom libraries of standards for targeted proteomics based on parallel reaction monitoring (PRM). The recombinant fragments can be produced in a bacterial cell factory to generate heavy isotope labeled standards for absolute quantification of the corresponding protein targets and be used to produce high- quality spectral libraries. Altogether, coordinates for 25,684 unique proteotypic peptide assays have been experimentally defined covering 10,163 human proteins. The protocol allows for precise monitoring of digestion kinetics and thus enables to select peptides that behave quantitative during the sample preparation process. We show that the quantification tag of each recombinant protein fragment can be used for accurate retention time prediction and allows for assay standardization across different method parameters. The use of this resource was illustrated by determining the absolute concentrations of selected protein targets using multiplex targeted proteomics assays for determination of quantitative assessment of 49 protein targets in serum samples.
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| 3. |
- Edfors, Fredrik, et al.
(författare)
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Immunoproteomics using polyclonal antibodies and stable isotope-labeled affinity-purified recombinant proteins
- 2014
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Ingår i: Molecular & Cellular Proteomics. - 1535-9476 .- 1535-9484. ; 13:6, s. 1611-1624
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Tidskriftsartikel (refereegranskat)abstract
- AThe combination of immuno-based methods and mass spectrometry detection has great potential in the field of quantitative proteomics. Here, we describe a new method (immuno-SILAC) for the absolute quantification of proteins in complex samples based on polyclonal antibodies and stable isotope-labeled recombinant protein fragments to allow affinity enrichment prior to mass spectrometry analysis and accurate quantification. We took advantage of the antibody resources publicly available from the Human Protein Atlas project covering more than 80% of all human protein-coding genes. Epitope mapping revealed that a majority of the polyclonal antibodies recognized multiple linear epitopes, and based on these results, a semi-automated method was developed for peptide enrichment using polyclonal antibodies immobilized on protein A-coated magnetic beads. A protocol based on the simultaneous multiplex capture of more than 40 protein targets showed that approximately half of the antibodies enriched at least one functional peptide detected in the subsequent mass spectrometry analysis. The approach was further developed to also generate quantitative data via the addition of heavy isotope-labeled recombinant protein fragment standards prior to trypsin digestion. Here, we show that we were able to use small amounts of antibodies (50 ng per target) in this manner for efficient multiplex analysis of quantitative levels of proteins in a human HeLa cell lysate. The results suggest that polyclonal antibodies generated via immunization of recombinant protein fragments could be used for the enrichment of target peptides to allow for rapid mass spectrometry analysis taking advantage of a substantial reduction in sample complexity. The possibility of building up a proteome-wide resource for immuno-SILAC assays based on publicly available antibody resources is discussed.
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| 4. |
- Edfors, Fredrik, et al.
(författare)
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Screening a Resource of Recombinant Protein Fragments for Targeted Proteomics
- 2019
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Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 18:7, s. 2706-2718
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Tidskriftsartikel (refereegranskat)abstract
- The availability of proteomics resources hosting protein and peptide standards, as well as the data describing their analytical performances, will continue to enhance our current capabilities to develop targeted proteomics methods for quantitative biology. This study describes the analysis of a resource of 26,840 individually purified recombinant protein fragments corresponding to more than 16,000 human protein-coding genes. The resource was screened to identify proteotypic peptides suitable for targeted proteomics efforts, and we report LC-MS/MS assay coordinates for more than 25,000 proteotypic peptides, corresponding to more than 10,000 unique proteins. Additionally, peptide formation and digestion kinetics were, for a subset of the standards, monitored using a time-course protocol involving parallel digestion of isotope-labeled recombinant protein standards and endogenous human plasma proteins. We show that the strategy by adding isotope-labeled recombinant proteins before trypsin digestion enables short digestion protocols (<= 60 min) with robust quantitative precision. In a proof-of-concept study, we quantified 23 proteins in human plasma using assay parameters defined in our study and used the standards to describe distinct clusters of individuals linked to different levels of LPA, APOE, SERPINAS, and TFRC. In summary, we describe the use and utility of a resource of recombinant proteins to identify proteotypic peptides useful for targeted proteomics assay development.
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| 5. |
- Eriksson, Anders I., et al.
(författare)
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Cold and warm electrons at comet 67P/Churyumov-Gerasimenko
- 2017
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Ingår i: Astronomy and Astrophysics. - : EDP SCIENCES S A. - 0004-6361 .- 1432-0746. ; 605
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Tidskriftsartikel (refereegranskat)abstract
- Context. Strong electron cooling on the neutral gas in cometary comae has been predicted for a long time, but actual measurements of low electron temperature are scarce. Aims. Our aim is to demonstrate the existence of cold electrons in the inner coma of comet 67P/Churyumov-Gerasimenko and show filamentation of this plasma. Methods. In situ measurements of plasma density, electron temperature and spacecraft potential were carried out by the Rosetta Langmuir probe instrument, LAP. We also performed analytical modelling of the expanding two-temperature electron gas. Results. LAP data acquired within a few hundred km from the nucleus are dominated by a warm component with electron temperature typically 5-10 eV at all heliocentric distances covered (1.25 to 3.83 AU). A cold component, with temperature no higher than about 0.1 eV, appears in the data as short (few to few tens of seconds) pulses of high probe current, indicating local enhancement of plasma density as well as a decrease in electron temperature. These pulses first appeared around 3 AU and were seen for longer periods close to perihelion. The general pattern of pulse appearance follows that of neutral gas and plasma density. We have not identified any periods with only cold electrons present. The electron flux to Rosetta was always dominated by higher energies, driving the spacecraft potential to order -10 V. Conclusions. The warm (5-10 eV) electron population observed throughout the mission is interpreted as electrons retaining the energy they obtained when released in the ionisation process. The sometimes observed cold populations with electron temperatures below 0.1 eV verify collisional cooling in the coma. The cold electrons were only observed together with the warm population. The general appearance of the cold population appears to be consistent with a Haser-like model, implicitly supporting also the coupling of ions to the neutral gas. The expanding cold plasma is unstable, forming filaments that we observe as pulses.
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