Screening a Resource of Recombinant Protein Fragments for Targeted Proteomics

Edfors, Fredrik; Forsström, Björn; Vunk, Helian; Kotol, David; Fredolini, Claudia; Maddalo, Gianluca; Svensson, Anne Sophie; Boström, Tove; Tegel, Hanna; Nilsson, Peter; Schwenk, Jochen M.; Uhlén, Mathias

doi:10.1021/acs.jproteome.8b00924

Cited by 19 publications

(30 citation statements)

References 37 publications

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“…Finally, we aimed to confirm the data obtained by the selected Ab pairs though using additional analyses. This assessment was based on comparing our data with results from targeted plasma MS analysis and solution‐based proximity extension assays (PEA) . For above methods, we obtained data sets generated in previous studies of the longitudinal sample analyzed in the application phase (Fagerberg et al., unpublished).…”

Section: Resultsmentioning

confidence: 99%

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Systematic Development of Sandwich Immunoassays for the Plasma Secretome

et al. 2019

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The plasma proteome offers a clinically useful window into human health. Recent advances from highly multiplexed assays now call for appropriate pipelines to validate individual candidates. Here, a workflow is developed to build dual binder sandwich immunoassays (SIA) and for proteins predicted to be secreted into plasma. Utilizing suspension bead arrays, ≈1800 unique antibody pairs are first screened against 209 proteins with recombinant proteins as well as EDTA plasma. Employing 624 unique antibodies, dilution‐dependent curves in plasma and concentration‐dependent curves of full‐length proteins for 102 (49%) of the targets are obtained. For 22 protein assays, the longitudinal, interindividual, and technical performance is determined in a set of plasma samples collected from 18 healthy subjects every third month over 1 year. Finally, 14 of these assays are compared with with SIAs composed of other binders, proximity extension assays, and affinity‐free targeted mass spectrometry. The workflow provides a multiplexed approach to screen for SIA pairs that suggests using at least three antibodies per target. This design is applicable for a wider range of targets of the plasma proteome, and the assays can be applied for discovery but also to validate emerging candidates derived from other platforms.

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Section: Resultsmentioning

confidence: 99%

“…For orthogonal comparison of the targets, the overlap of the chosen 21 targets was correlated with data achieved by two independent experimental setups for the same sample selection: 1) a recently published targeted MS approach and 2) multiplex proximity extension assays (Olink Bioscience, Uppsala Sweden) …”

Section: Methodsmentioning

confidence: 99%

Systematic Development of Sandwich Immunoassays for the Plasma Secretome

et al. 2019

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“…Lastly, we aimed to confirm the data obtained by the selected Ab pairs though using additional analyses. This assessment was based on comparing our data with results from targeted plasma MS analysis [8] and solution-based proximity extension assays (PEA) [9]. For above methods, we obtained data sets generated in previous studies of the longitudinal sample analyzed in the application phase (Fagerberg et al, unpublished).…”

Section: Comparison To Orthogonal Plasma Assaysmentioning

confidence: 99%

“…(1) a recently published targeted MS approach [8] and (2) multiplex proximity extension assays (Olink Bioscience, Uppsala Sweden) [9].…”

Section: Orthogonal Assaysmentioning

confidence: 99%

“…After washing off unbound proteins, the captured proteins were detected via biotinylated antibodies (6)(7). The readout occurred by the addition of a streptavidinfluorophore and using the Luminex systems (8). Protein concentration achieved from the SIA pairs was compared with data from targeted plasma mass spectrometry analysis and solution-based proximity extension assays, which was generated on the same sample set.…”

Section: Additional Informationmentioning

confidence: 99%

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Systematic Development of Sandwich Immunoassays for the Plasma Secretome

Haeussler

Bendes

Iglesias

et al. 2019

Preprint

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The plasma proteome offers a clinically useful window into human health and disease. With recent progress made on the development of highly multiplexed immunoassays with high sample throughput, a remaining need is to establish a pipeline for validating the individual proteins that build such bio-signatures by using targeted assays. In order to streamline such efforts, we developed a workflow to build dual binder sandwich immunoassays (SIA) and chose to evaluate this on proteins predicted to be secreted form cells and tissues. Utilizing the multiplexing capacities of the bead array technology, we first screened ~ 1,800 unique antibody pairs against 209 protein targets and collected data from dilution series of recombinant proteins as well as EDTA plasma. Employing 624 unique antibodies from the Human Protein Atlas, we obtained dilution-dependent curves in plasma and concentration-dependent curves of fulllength proteins for 102 (49%) of the targets. For 22 protein assays, the longitudinal, interindividual and technical performance was determined in a set of plasma samples collected from 18 healthy subjects every third month over one year. Lastly, we compared 14 of these assays with SIAs composed of other binders, proximity extension assays and affinity-free targeted mass spectrometry. Our workflow provides a multiplexed approach to screen for SIA pairs that suggests using at least three antibodies per target. This design is applicable for a wider range of targets of the plasma proteome, while the assays can be applied for discovery but also to validate emerging candidates derived from other platforms.(245 words) knowledge or particular interest but rather availability of reagents to conduct this proof-ofconcept study from screening, via validation to the analysis of longitudinal samples.

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Detailed Method for Performing the ExSTA Approach in Quantitative Bottom-Up Plasma Proteomics

Percy

Borchers

2021

Methods in Molecular Biology

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The use of stable isotope-labeled standards (SIS) is an analytically valid means of quantifying proteins in biological samples. The nature of the labeled standards and their point of insertion in a bottom-up proteomic workflow can vary, with quantification methods utilizing curves in analytically sound practices. A promising quantification strategy for low sample amounts is external standard addition (ExSTA). In ExSTA, multipoint calibration curves are generated in buffer using serially diluted natural (NAT) peptides and a fixed concentration of SIS peptides. Equal concentrations of SIS peptides are spiked into experimental sample digests, with all digests (control and experimental) subjected to solid-phase extraction prior to liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis. Endogenous peptide concentrations are then determined using the regression equation of the standard curves. Given the benefits of ExSTA in large-scale analysis, a detailed protocol is provided herein for quantifying a multiplexed panel of 125 high-to-moderate abundance proteins in undepleted and non-enriched human plasma samples. The procedural details and recommendations for successfully executing all phases of this quantification approach are described. As the proteins have been putatively correlated with various noncommunicable diseases, quantifying these by ExSTA in large-scale studies should help rapidly and precisely assess their true biomarker efficacy.

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Screening a Resource of Recombinant Protein Fragments for Targeted Proteomics

Cited by 19 publications

References 37 publications

Systematic Development of Sandwich Immunoassays for the Plasma Secretome

Systematic Development of Sandwich Immunoassays for the Plasma Secretome

Systematic Development of Sandwich Immunoassays for the Plasma Secretome

Detailed Method for Performing the ExSTA Approach in Quantitative Bottom-Up Plasma Proteomics

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