Multiplexed protein profiling by sequential affinity capture

Ayoglu, Burcu; Birgersson, Elin; Mezger, Anja; Nilsson, Mats; Uhlén, Mathias; Nilsson, Peter; Schwenk, Jochen M.

doi:10.1002/pmic.201500398

Cited by 7 publications

(4 citation statements)

References 19 publications

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“…Each bar-coded bead population is coupled to a specific antibody (Ab), allowing the parallel capture and detection of multiple unique, identifiable protein epitopes from a complex mixture of proteins in solution. The SBA technology in combination with the Human Protein Atlas (HPA) Ab collection has been successfully implemented to identify soluble proteins in human serum samples (17,18). Here, we demonstrate an experimental approach to define the secretin-like GPCR-RAMP interactome and develop an assay capable of testing the hypothesis that GPCRs and RAMPs interact on a global level.…”

Section: Introductionmentioning

confidence: 99%

Multiplexed analysis of the secretin-like GPCR-RAMP interactome

et al. 2019

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Receptor activity–modifying proteins (RAMPs) have been shown to modulate the functions of several G protein–coupled receptors (GPCRs), but potential direct interactions among the three known RAMPs and hundreds of GPCRs have never been investigated. Focusing mainly on the secretin-like family of GPCRs, we engineered epitope-tagged GPCRs and RAMPs, and developed a multiplexed suspension bead array (SBA) immunoassay to detect GPCR-RAMP complexes from detergent-solubilized lysates. Using 64 antibodies raised against the native proteins and 4 antibodies targeting the epitope tags, we mapped the interactions among 23 GPCRs and 3 RAMPs. We validated nearly all previously reported secretin-like GPCR-RAMP interactions, and also found previously unidentified RAMP interactions with additional secretin-like GPCRs, chemokine receptors, and orphan receptors. The results provide a complete interactome of secretin-like GPCRs with RAMPs. The SBA strategy will be useful to search for additional GPCR-RAMP complexes and other interacting membrane protein pairs in cell lines and tissues.

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Section: Introductionmentioning

confidence: 99%

Multiplexed analysis of the secretin-like GPCR-RAMP interactome

et al. 2019

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“…Each bar-coded bead population is coupled to a specific antibody (Ab), allowing the parallel capture and detection of multiple unique, identifiable protein epitopes from a complex mixture of proteins in solution. The SBA technology in combination with the Human Protein Atlas (HPA) Ab collection has been successfully implemented to identify soluble proteins in human serum samples (16, 17) . Here we demonstrate an experimental approach to define the secretin-like GPCR-RAMP interactome, and develop an assay capable of testing the hypothesis that GPCRs and RAMPs interact on a global level.…”

Section: Introductionmentioning

confidence: 99%

Multiplexed Analysis of the Secretin-like GPCR-RAMP Interactome

Lorenzen

Dodig‐Crnković

Kotliar

et al. 2019

Preprint

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20Although receptor activity-modifying proteins (RAMPs) have been shown to modulate the 21 functions of several different G protein-coupled receptors (GPCRs), potential direct interactions 22 among the three known RAMPs and hundreds of GPCRs has never been investigated. We 23 engineered three epitope-tagged RAMPs and 23 epitope-tagged GPCRs, focusing on the secretin-24 like family of GPCRs, and developed a suspension bead array (SBA) immunoassay designed to 25 detect RAMP-GPCR complexes. We then used 64 antibodies raised against native RAMPs and 26 GPCRs, along with four antibodies targeting the epitope tags, to multiplex the SBA assay to 27 detect and measure all possible combinations of interaction among the 23 GPCRs and three 28 RAMPs. The results of the SBA assay provide a complete interactome of secretin-like GPCRs 29 with RAMPs. We demonstrate direct interaction of previously reported secretin-like GPCRs 30 whose functions are modulated by RAMPs. We also discovered novel sets of GPCR-RAMP 31 interacting pairs, and found additional secretin-like GPCRs, chemokine receptors and orphan 32 receptors that interact with RAMPs. Using in situ proximity ligation assay, we verified a subset of 33 these novel GPCR-RAMP interactions in cell membranes. In total, we found GPCR-RAMP 34 interactions for the majority of the 23 GPCRs tested. Each GPCR interacted with either all three 35 RAMPs or with RAMP2 and RAMP3, with the exception of one GPCR that interacted with just 36 RAMP3. In summary, we describe an SBA strategy that will be useful to search for GPCR-37 RAMP interactions in cell lines and tissues, and conclude that GPCR-RAMP interactions are 38 more common than previously appreciated. 39 40 42 commonly targeted for drug development. Emerging evidence suggests that the surface expression 43 and activity of GPCRs can be modulated by receptor activity-modifying proteins (RAMPs), a 44 ubiquitously-expressed family of single-pass membrane proteins with only three members. First discovered as a necessary component for the function of calcitonin-like receptor (CALCRL), 46 RAMPs were shown to alter ligand specificity of both CALCRL and calcitonin receptor (CALCR) 47 by dictating to which ligand they respond , (1-3). Interaction of RAMPs with GPCRs has since been 48 demonstrated with several additional members of the secretin-like GPCRs. RAMPs can influence 49 several features of GPCR biology, including trafficking to the cell membrane, ligand specificity, 50 downstream signaling and recycling (4-11). However, many of the secretin-like GPCR-RAMP 51 interactions remain unverified, and most of the interactions have not been demonstrated using a 52 direct binding assay. In addition, prior reports show conflicting results regarding whether certain 53 secretin-like GPCRs form complexes with RAMPs. 54There are also suggestions that RAMPs form complexes with a broader set of GPCRs. A 55 single GPCR from the glutamate-like family, calcium-sensing receptor (CaSR), and a single GPCR 56 from the rhodopsin-like family, G protein-coup...

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“…In fact, to overcome the above problems and challenges, researchers have continued to improve protein microarrays. Protein microarrays with higher performance, such as high‐sensitivity antibody microarrays [110], multiplex glycan bead arrays [111] and three‐dimensionally functionalised reverse‐phase glycoprotein arrays (GPA) [112], have been developed.…”

Section: Perspectivesmentioning

confidence: 99%

Research progress in protein microarrays: Focussing on cancer research

Chen

Yang

Liu

et al. 2022

Proteomics Clinical Apps

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Although several effective treatment modalities have been developed for cancers, the morbidity and mortality associated with cancer continues to increase every year. As one of the most exciting emerging technologies, protein microarrays represent a powerful tool in the field of cancer research because of their advantages such as high throughput, small sample usage, more flexibility, high sensitivity and direct readout of results. In this review, we focus on the research progress in four types of protein microarrays (proteome microarray, antibody microarray, lectin microarray and reversed protein array) with emphasis on their application in cancer research. Finally, we discuss the current challenges faced by protein microarrays and directions for future developments. We firmly believe that this novel systems biology research tool holds immense potential in cancer research and will become an irreplaceable tool in this field.

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Multiplexed protein profiling by sequential affinity capture

Cited by 7 publications

References 19 publications

Multiplexed analysis of the secretin-like GPCR-RAMP interactome

Multiplexed analysis of the secretin-like GPCR-RAMP interactome

Multiplexed Analysis of the Secretin-like GPCR-RAMP Interactome

Research progress in protein microarrays: Focussing on cancer research

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