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ACS Chemical Biology Apr 2023Cell surface proteins (CSPs) are vital molecular mediators for cells and their extracellular environment. Thus, understanding which CSPs are displayed on cells,...
Cell surface proteins (CSPs) are vital molecular mediators for cells and their extracellular environment. Thus, understanding which CSPs are displayed on cells, especially in different cell states, remains an important endeavor in cell biology. Here, we describe the integration of cell surface engineering with radical-mediated protein biotinylation to profile CSPs. This method relies on the prefunctionalization of cells with cholesterol lipid groups, followed by sortase-catalyzed conjugation with an APEX2 ascorbate peroxidase enzyme. In the presence of biotin-phenol and HO, APEX2 catalyzes the formation of highly reactive biotinyl radicals that covalently tag electron-rich residues within CSPs for subsequent streptavidin-based enrichment and analysis by quantitative mass spectrometry. While APEX2 is traditionally used to capture proximity-based interactomes, we envisioned using it in a "baitless" manner on cell surfaces to capture CSPs. We evaluate this strategy in light of another CSP labeling method that relies on the presence of cell surface sialic acid. Using the APEX2 strategy, we describe the CSPs found in three mammalian cell lines and compare CSPs in adherent versus three-dimensional pancreatic adenocarcinoma cells.
Topics: Animals; Humans; Adenocarcinoma; Biotinylation; Cell Membrane; Hydrogen Peroxide; Mammals; Membrane Proteins; Pancreatic Neoplasms; Streptavidin; Proteomics
PubMed: 35443134
DOI: 10.1021/acschembio.1c00865 -
Interrogating Kinase-Substrate Relationships with Proximity Labeling and Phosphorylation Enrichment.Journal of Proteome Research Feb 2022Kinases govern many cellular responses through the reversible transfer of a phosphate moiety to their substrates. However, pairing a substrate with a kinase is...
Kinases govern many cellular responses through the reversible transfer of a phosphate moiety to their substrates. However, pairing a substrate with a kinase is challenging. In proximity labeling experiments, proteins proximal to a target protein are marked by biotinylation, and mass spectrometry can be used for their identification. Here, we combine ascorbate peroxidase (APEX) proximity labeling and a phosphorylation enrichment-based workflow, Phospho-APEX (pAPEX), to rapidly identify phosphorylated and biotinylated neighbor proteins which can be considered for candidate substrates. The pAPEX strategy enriches and quantifies differences in proximity for proteins and phosphorylation sites proximal to an APEX2-tagged kinase under the kinase "ON" and kinase "OFF" conditions. As a proof of concept, we identified candidate substrates of MAPK1 in HEK293T and HCT116 cells and candidate substrates of PKA in HEK293T cells. In addition to many known substrates, C15orf39 was identified and confirmed as a novel MAPK1 substrate. In all, we adapted the proximity labeling-based platform to accommodate phosphorylation analysis for kinase substrate identification.
Topics: Ascorbate Peroxidases; Biotinylation; HEK293 Cells; Humans; Mass Spectrometry; Phosphorylation; Substrate Specificity
PubMed: 35044772
DOI: 10.1021/acs.jproteome.1c00865 -
STAR Protocols Dec 2022Analysis of the surfaceome of a blood cell subset requires cell sorting, followed by surface protein enrichment. Here, we present a protocol combining magnetically...
Analysis of the surfaceome of a blood cell subset requires cell sorting, followed by surface protein enrichment. Here, we present a protocol combining magnetically activated cell sorting (MACS) and surface biotinylation of the target cell subset from human peripheral blood mononuclear cells (PBMCs). We describe the steps for isolating target cells and their in-column surface biotinylation, followed by isolation and mass spectrometry analysis of biotinylated proteins. The protocol enables in-column surface biotinylation of specific cell subsets with minimal membrane disruption.
Topics: Humans; Biotinylation; Leukocytes, Mononuclear; Cell Membrane; Membrane Proteins; Magnetic Phenomena
PubMed: 36595912
DOI: 10.1016/j.xpro.2022.101863 -
Nature Communications Jan 2020Axon initial segments (AISs) generate action potentials and regulate the polarized distribution of proteins, lipids, and organelles in neurons. While the mechanisms of...
Axon initial segments (AISs) generate action potentials and regulate the polarized distribution of proteins, lipids, and organelles in neurons. While the mechanisms of AIS Na and K channel clustering are understood, the molecular mechanisms that stabilize the AIS and control neuronal polarity remain obscure. Here, we use proximity biotinylation and mass spectrometry to identify the AIS proteome. We target the biotin-ligase BirA* to the AIS by generating fusion proteins of BirA* with NF186, Ndel1, and Trim46; these chimeras map the molecular organization of AIS intracellular membrane, cytosolic, and microtubule compartments. Our experiments reveal a diverse set of biotinylated proteins not previously reported at the AIS. We show many are located at the AIS, interact with known AIS proteins, and their loss disrupts AIS structure and function. Our results provide conceptual insights and a resource for AIS molecular organization, the mechanisms of AIS stability, and polarized trafficking in neurons.
Topics: Animals; Axon Initial Segment; Axons; Biotinylation; Humans; Mass Spectrometry; Mice; Neurons; Protein Transport; Proteome; Rats; Rats, Sprague-Dawley
PubMed: 31900387
DOI: 10.1038/s41467-019-13658-5 -
Molecular Systems Biology May 2020Streptavidin-mediated enrichment is a powerful strategy to identify biotinylated biomolecules and their interaction partners; however, intense streptavidin-derived...
Streptavidin-mediated enrichment is a powerful strategy to identify biotinylated biomolecules and their interaction partners; however, intense streptavidin-derived peptides impede protein identification by mass spectrometry. Here, we present an approach to chemically modify streptavidin, thus rendering it resistant to proteolysis by trypsin and LysC. This modification results in over 100-fold reduction of streptavidin contamination and in better coverage of proteins interacting with various biotinylated bait molecules (DNA, protein, and lipid) in an overall simplified workflow.
Topics: Arginine; Biotinylation; Chromatin Immunoprecipitation; HeLa Cells; Humans; Lysine; Mass Spectrometry; Membrane Proteins; Metalloendopeptidases; Neoplasm Proteins; Polycomb Repressive Complex 2; Proteins; Proteolysis; Proteomics; Streptavidin; Transcription Factors; Trypsin
PubMed: 32400114
DOI: 10.15252/msb.20199370 -
MSphere Jun 2023The flagellated kinetoplastid protozoan and causative agent of human Chagas disease, Trypanosoma cruzi, inhabits both invertebrate and mammalian hosts over the course of...
The flagellated kinetoplastid protozoan and causative agent of human Chagas disease, Trypanosoma cruzi, inhabits both invertebrate and mammalian hosts over the course of its complex life cycle. In these disparate environments, T. cruzi uses its single flagellum to propel motile life stages and, in some instances, to establish intimate contact with the host. Beyond its role in motility, the functional capabilities of the T. cruzi flagellum have not been defined. Moreover, the lack of proteomic information for this organelle, in any parasite life stage, has limited functional investigation. In this study, we employed a proximity-dependent biotinylation approach based on the differential targeting of the biotin ligase TurboID to the flagellum or cytosol in replicative stages of T. cruzi to identify proteins that are enriched in the flagellum by mass spectrometry. Proteomic analysis of the resulting biotinylated protein fractions yielded 218 candidate flagellar proteins in T. cruzi epimastigotes (insect stage) and 99 proteins in intracellular amastigotes (mammalian stage). Forty of these enriched flagellar proteins were common to both parasite life stages and included orthologs of known flagellar proteins in other trypanosomatid species, proteins specific to the T. cruzi lineage and hypothetical proteins. With the validation of flagellar localization for several of the identified candidates, our results demonstrate that TurboID-based proximity proteomics is an effective tool for probing subcellular compartments in T. cruzi. The proteomic data sets generated in this work offer a valuable resource to facilitate functional investigation of the understudied T. cruzi flagellum. Trypanosoma cruzi is a protozoan parasite that causes Chagas disease, which causes substantial morbidity and mortality in South and Central America. Throughout its life cycle, T. cruzi interacts with insect and mammalian hosts via its single flagellum, establishing intimate contact with host membranes. Currently, few flagellar proteins have been identified in T. cruzi that could provide insight into the mechanisms involved in mediating physical and biochemical interactions with the host. Here, we set out to identify flagellar proteins in the main replicative stages of T. cruzi using a proximity-labeling approach coupled with mass spectrometry. The >200 candidate flagellar proteins identified represent the first large-scale identification of candidate flagellar proteins in T. cruzi with preliminary validation. These data offer new avenues to investigate the biology of T. cruzi-host interactions, a promising area for development of new strategies aimed at the control of this pathogen.
Topics: Animals; Humans; Trypanosoma cruzi; Biotinylation; Proteomics; Chagas Disease; Protozoan Proteins; Mammals
PubMed: 37017578
DOI: 10.1128/msphere.00088-23 -
Journal of the American Society For... Feb 2020The use of biotin or biotin-containing reagents is an essential component of many protein purification and labeling technologies. Owing to its small size and high...
The use of biotin or biotin-containing reagents is an essential component of many protein purification and labeling technologies. Owing to its small size and high affinity to the avidin family of proteins, biotin is a versatile molecular handle that permits both enrichment and purity that is not easily achieved by other reagents. Traditionally, the use of biotinylation to enrich for proteins has not required the detection of the site of biotinylation. However, newer technologies for discovery of protein-protein interactions, such as APEX and BioID, as well as some of the click chemistry-based labeling approaches have underscored the importance of determining the exact residue that is modified by biotin. Anti-biotin antibody-based enrichment of biotinylated peptides (e.g., BioSITe) coupled to LC-MS/MS permit large-scale detection and localization of sites of biotinylation. As with any chemical modification of peptides, understanding the fragmentation patterns that result from biotin modification is essential to improving its detection by LC-MS/MS. Tandem mass spectra of biotinylated peptides has not yet been studied systematically. Here, we describe the various signature fragment ions generated with collision-induced dissociation of biotinylated peptides. We focused on biotin adducts attached to peptides generated by BioID and APEX experiments, including biotin, isotopically heavy biotin, and biotin-XX-phenol, a nonpermeable variant of biotin-phenol. We also highlight how the detection of biotinylated peptides in high-throughput studies poses certain computational challenges for accurate quantitation which need to be addressed. Our findings about signature fragment ions of biotinylated peptides should be helpful in the confirmation of biotinylation sites.
Topics: Amino Acid Sequence; Animals; Biotin; Biotinylation; Cattle; Ions; Lysine; Peptides; Serum Albumin, Bovine; Tandem Mass Spectrometry; Tyrosine
PubMed: 31939678
DOI: 10.1021/jasms.9b00024 -
Frontiers in Cellular and Infection... 2022Proximity labelling is a powerful and rapidly developing technology for exploring the interaction space and molecular environment of a protein of interest at the... (Review)
Review
Proximity labelling is a powerful and rapidly developing technology for exploring the interaction space and molecular environment of a protein of interest at the nanometre scale. In proximity labelling, a promiscuous biotinylating enzyme is genetically fused to the protein of interest, initiation of labelling then results in the biotinylating enzyme generating reactive biotin which covalently 'tags' nearby molecules. Importantly, this labelling takes place whilst the protein of interest continues to perform its normal functions in the cell. Due to its unique advantageous characteristics, proximity labelling is driving discoveries in an ever increasing range of organisms. Here, we highlight the applications of proximity labelling to the study of kinetoplastids, a group of eukaryotic protozoa that includes trypanosomes and which can cause serious disease in humans and livestock. We first provide a general overview of the proximity labelling experimental workflow including key labelling enzymes used, proper experimental design with appropriate controls and robust statistical analysis to maximise the amount of reliable spatial information that is generated. We discuss studies employing proximity labelling in kinetoplastid parasites to illustrate how these key principles of experimental design are applied. Finally, we highlight emerging trends in the development of proximity labelling methodology.
Topics: Animals; Biotin; Biotinylation; Humans; Kinetoplastida; Parasites
PubMed: 35601102
DOI: 10.3389/fcimb.2022.894213 -
ACS Pharmacology & Translational Science Feb 2022BirA*, a mutant form of the biotinylating enzyme BirA, can nonspecifically biotinylate ε-amino groups on lysines of proteins. Based on the promiscuous labeling nature...
BirA*, a mutant form of the biotinylating enzyme BirA, can nonspecifically biotinylate ε-amino groups on lysines of proteins. Based on the promiscuous labeling nature of BirA*, plasmids expressing fusion constructs of BirA* to a given ligand have been used to transfect eukaryotic cells, leading to the biotinylation of intracellular proteins interacting or in close proximity to such Ligand.BirA* constructs. Mass spectrometry performed on the recovered biotinylated partners allows one to map intracellular protein interactors, a technique known as BioID. In contrast, the expression and purification of recombinant Ligand.BirA* constructs could serve as a powerful tool for labeling and detecting cell surface receptors. Here, we report the design and expression of recombinant Affibody.BirA* constructs, Z.BirA* and Z.BirA*, as protein bispecifics able to biotinylate their respective receptors EGFR and HER2 on the surface of MDA-MB-231 (EGFR, EpCaM, and CD44) and SK-OV-3 (HER2, EGFR, EpCaM, and CD44) cancer cells. These Affibody.BirA* constructs retain both their BirA* enzymatic activity as well as their receptor-binding function. Importantly, MDA-MB-231 and SK-OV-3 cells biotinylated with Affibody.BirA* constructs did label their receptors EGFR and HER2 but did not biotinylate irrelevant antigens such as EpCaM or CD44 present on the surface of both cell lines. Ligand.BirA* bispecifics may represent a promising class of agents to identify unknown receptors on cell surfaces.
PubMed: 36742360
DOI: 10.1021/acsptsci.1c00192 -
Molecular & Cellular Proteomics : MCP Jul 2022Identifying protein-protein and other proximal interactions is central to dissecting signaling and regulatory processes in cells. BioID is a proximity-dependent...
Identifying protein-protein and other proximal interactions is central to dissecting signaling and regulatory processes in cells. BioID is a proximity-dependent biotinylation method that uses an "abortive" biotin ligase to detect proximal interactions in cells in a highly reproducible manner. Recent advancements in proximity-dependent biotinylation tools have improved efficiency and timing of labeling, allowing for measurement of interactions on a cellular timescale. However, issues of size, stability, and background labeling of these constructs persist. Here we modified the structure of BioID2, derived from Aquifex aeolicus BirA, to create a smaller, highly active, biotin ligase that we named MicroID2. Truncation of the C terrminus of BioID2 and addition of mutations to alleviate blockage of biotin/ATP binding at the active site of BioID2 resulted in a smaller and highly active construct with lower background labeling. Several additional point mutations improved the function of our modified MicroID2 construct compared with BioID2 and other biotin ligases, including TurboID and miniTurbo. MicroID2 is the smallest biotin ligase reported so far (180 amino acids [AAs] for MicroID2 versus 257 AAs for miniTurbo and 338 AAs for TurboID), yet it demonstrates only slightly less labeling activity than TurboID and outperforms miniTurbo. MicroID2 also had lower background labeling than TurboID. For experiments where precise temporal control of labeling is essential, we in addition developed a MicroID2 mutant, termed lbMicroID2 (low background MicroID2), that has lower labeling efficiency but significantly reduced biotin scavenging compared with BioID2. Finally, we demonstrate utility of MicroID2 in mass spectrometry experiments by localizing MicroID2 constructs to subcellular organelles and measuring proximal interactions.
Topics: Biotin; Biotinylation; Ligases; Mass Spectrometry; Protein Interaction Mapping; Proteomics
PubMed: 35688383
DOI: 10.1016/j.mcpro.2022.100256